Antibodies to IL-17A

ABSTRACT

Engineered antibodies to human IL-17A are provided, as well as uses thereof.

The present application is a divisional of Ser. No. 11/836,318, filedAug. 9, 2007, now U.S. Pat. No. 7,846,443, which claims the benefit ofU.S. Provisional Patent Application No. 60/837,197, filed Aug. 11, 2006,which is incorporated by reference.

FIELD OF THE INVENTION

The present invention relates generally to IL-17A specific bindingcompounds, such as antibodies, and uses thereof. More specifically, theinvention relates to chimeric and humanized antibodies that recognizehuman IL-17A and modulate its activity, particularly in inflammatory,autoimmune and proliferative disorders.

BACKGROUND OF THE INVENTION

The immune system functions to protect individuals from infectiveagents, e.g., bacteria, multi-cellular organisms, and viruses, as wellas from cancers. This system includes several types of lymphoid andmyeloid cells such as monocytes, macrophages, dendritic cells (DCs),eosinophils, T cells, B cells, and neutrophils. These lymphoid andmyeloid cells often produce signaling proteins known as cytokines. Theimmune response includes inflammation, i.e., the accumulation of immunecells systemically or in a particular location of the body. In responseto an infective agent or foreign substance, immune cells secretecytokines which, in turn, modulate immune cell proliferation,development, differentiation, or migration. Immune response can producepathological consequences, e.g., when it involves excessiveinflammation, as in the autoimmune disorders (see, e.g., Abbas et al.(eds.) (2000) Cellular and Molecular Immunology, W.B. Saunders Co.,Philadelphia, Pa.; Oppenheim and Feldmann (eds.) (2001) CytokineReference, Academic Press, San Diego, Calif.; von Andrian and Mackay(2000) New Engl. J. Med. 343:1020-1034; Davidson and Diamond (2001) NewEngl. J. Med. 345:340-350).

Interleukin-17A (IL-17A; also known as Cytotoxic T-Lymphocyte-associatedAntigen 8 (CTLA8), IL-17) is a homodimeric cytokine produced by memory Tcells following antigen recognition. The development of such T cells ispromoted by interleukin-23 (IL-23). McKenzie et al. (2006) TrendsImmunol. 27(1): 17-23; Langrish et al. (2005) J. Exp. Med.201(2):233-40. IL-17A acts through two receptors, IL-17RA and IL-17RC toinduce the production of numerous molecules involved in neutrophilbiology, inflammation, and organ destruction. This cytokine synergizeswith tissue necrosis factor (TNF) and or interleukin 1β (IL-1β) topromote a greater pro-inflammatory environment. Antagonizing theactivity of IL-17A with antibodies or antigen binding fragments ofantibodies has been proposed for the treatment of a variety ofinflammatory, immune and proliferative disorders, including rheumatoidarthritis (RA), osteoarthritis, rheumatoid arthritis osteoporosis,inflammatory fibrosis (e.g. scleroderma, lung fibrosis, and cirrhosis),gingivitis, periodontitis or other inflammatory periodontal diseases,inflammatory bowel disorders (e.g. Crohn's disease, ulcerative colitisand inflammatory bowel disease), asthma (including allergic asthma),allergies, chronic obstructive pulmonary disease (COPD), multiplesclerosis, psoriasis and cancer. (See, e.g., US 2003/0166862, WO2005/108616, WO 2005/051422, and WO 2006/013107).

The most significant limitation in using antibodies as a therapeuticagent in vivo is the immunogenicity of the antibodies. As mostmonoclonal antibodies are derived from rodents, repeated use in humansresults in the generation of an immune response against the therapeuticantibody, e.g., human against mouse antibodies or HAMA. Such an immuneresponse results in a loss of therapeutic efficacy at a minimum and apotential fatal anaphylactic response at a maximum. Initial efforts toreduce the immunogenicity of rodent antibodies involved the productionof chimeric antibodies, in which mouse variable regions (Fv) were fusedwith human constant regions. Liu et al. (1987) Proc. Natl. Acad. Sci.USA 84:3439-43. However, mice injected with hybrids of human variableregions and mouse constant regions develop a strong anti-antibodyresponse directed against the human variable region, suggesting that theretention of the entire rodent Fv region in such chimeric antibodies maystill result in unwanted immunogenicity in patients.

It is generally believed that complementarity determining region (CDR)loops of variable domains comprise the binding site of antibodymolecules. Therefore, the grafting of rodent CDR loops onto humanframeworks (i.e., humanization) has been attempted to further minimizerodent sequences. Jones et al. (1986) Nature 321:522; Verhoeyen et al.(1988) Science 239:1534. However, CDR loop exchanges still do notuniformly result in an antibody with the same binding properties as theantibody of origin. Changes in framework residues (FR), residuesinvolved in CDR loop support, in humanized antibodies also are oftenrequired to preserve antigen binding affinity. Kabat et al. (1991) J.Immunol. 147:1709. While the use of CDR grafting and framework residuepreservation in a number of humanized antibody constructs has beenreported, it is difficult to predict if a particular sequence willresult in the antibody with the desired binding, and sometimesbiological, properties. See, e.g., Queen et al. (1989) Proc. Natl. Acad.Sci. USA 86:10029, Gorman et al. (1991) Proc. Natl. Acad. Sci. USA88:4181, and Hodgson (1991) Biotechnology (NY) 9:421-5. Moreover, mostprior studies used different human sequences for animal light and heavyvariable sequences, rendering the predictive nature of such studiesquestionable. Sequences of known antibodies have been used or, moretypically, those of antibodies having known X-ray crystal structures,such as antibodies NEW and KOL. See, e.g., Jones et al., supra;Verhoeyen et al., supra; and Gorman et al., supra. Exact sequenceinformation has been reported for a few humanized constructs.

The need exists for antagonists of IL-17A, such as anti-IL-17Amonoclonal antibodies, for use in treatment of human disorders, such asinflammatory, autoimmune, and proliferative disorders. Such antagonistswill preferably exhibit low immunogenicity in human subjects, allowingfor repeated administration without adverse immune responses.

SUMMARY OF THE INVENTION

The present invention relates to anti-human IL-17A antibodies having oneor more desirable properties, including high binding affinities,neutralizing activities, good pharmacokinetics and low antigenicity inhuman subjects. The invention also relates to use of the antibodies ofthe present invention in the treatment of disease.

Accordingly, in one embodiment the present invention provides a bindingcompound, for example an antibody molecule or binding fragment thereof,which binds human IL-17A and inhibits its activity. In some embodiments,the binding compound comprises at least one antibody light chainvariable (V_(L)) domain and at least one antibody heavy chain variable(V_(H)) domain, or binding fragments of these domains, wherein the V_(L)domain comprises at least a specified number of complementaritydetermining regions (CDRs) having a sequence selected from SEQ ID NOs:11-13, and the V_(H) domain comprises at least at least a specifiednumber of CDRs having a sequence selected from SEQ ID NOs:14-20, whereinthe specified number is one, two or three. The specified number of CDRsmay be the same or different for the light and heavy chain variabledomains in any given binding compound. In another embodiment, the V_(H)domain CDRs are selected from SEQ ID NOs:14, 17 and 20. In yet anotherembodiment, the V_(H) domain CDRs are selected from SEQ ID NOs:14, 16and 19. In a further embodiment, the sequences of the V_(L) and V_(H)domains are the sequences of SEQ ID NOs: 5 and 6, respectively. In someembodiments, the IL-17A binding compound inhibits the activity of humanIL-17A.

In other embodiments, the binding compound comprises at least one V_(L)domain and at least one V_(H) domain, or binding fragments of thesedomains, wherein the V_(L) domain comprises one, two or three CDRshaving a sequence selected from SEQ ID NOs: 26-28, and the V_(H) domaincomprises one, two or three CDRs having a sequence selected from SEQ IDNOs: 29-31. In another embodiment, the sequence of the V_(L) and V_(H)domains are the sequences of SEQ ID NOs: 22 and 23, respectively. Inanother embodiment, the binding compound has the same CDRs as theantibody produced from the hybridoma having ATCC Accession No. PTA-7739(rat 30C10, deposited as strain M7-30C10.C3 on Jul. 20, 2006).

In a further embodiment, the binding compound comprises at least oneV_(L) domain and at least one V_(H) domain, or binding fragments ofthese domains, wherein the V_(L) domain comprises one, two or three CDRshaving a sequence selected from SEQ ID NOs: 48-50, and the V_(H) domaincomprises one, two or three CDRs having a sequence selected from SEQ IDNOs: 51-53.

In yet other embodiments, the binding compound comprises at least oneV_(L) domain and at least one V_(H) domain, or binding fragments ofthese domains, wherein the V_(L) domain comprises one, two or three CDRshaving a sequence selected from SEQ ID NOs: 34-36, and the V_(H) domaincomprises one, two or three CDRs having a sequence selected from SEQ IDNOs: 37-39, or the V_(L) domain comprises one, two or three CDRs havinga sequence selected from SEQ ID NOs: 56-58, and the V_(H) domaincomprises one, two or three CDRs having a sequence selected from SEQ IDNOs: 59-61.

In various other embodiments, the present invention provides a bindingcompound that binds to human IL-17A that has V_(L) and V_(H) domainswith at least 95%, 90%. 85%, 80%, 75% or 50% sequence homology with thesequences of SEQ ID NOs: 5 and 6, respectively. In other embodiments thebinding compound of the present invention comprises V_(L) and V_(H)domains having up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreconservative amino acid substitutions with reference to the sequences ofSEQ ID NOs: 5 and 6, respectively. In another embodiment, the bindingcompound of the present invention is an antibody having a light chainand a heavy chain with up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreconservative amino acid substitutions with reference to the mature formsof the sequences of SEQ ID NOs: 2 and 4, respectively.

In one embodiment, the binding compound is an antibody or bindingfragment thereof, e.g. an antibody fragment selected from the groupconsisting of Fab, Fab′, Fab′-SH, Fv, scFv, F(ab′)₂, and a diabody. Inone embodiment, the binding compound of the present invention isantibody hu16C10 comprising a light chain having the sequence of themature form of SEQ ID NO.: 2 (residues 1-220) and a heavy chain havingthe sequence of the mature form of SEQ ID NO.: 4 (residues 1-454). Inanother embodiment, the binding compound is the antibody produced fromthe expression vector having ATCC Accession No. PTA-7675 (hu16C10 inplasmid pAIL17AV1, deposited Jun. 28, 2006).

In one embodiment, the binding compound of the present inventioncomprises a heavy chain constant region, for example a human constantregion, such as γ1, γ2, γ3, or γ4 human heavy chain constant region or avariant thereof. In another embodiment, the binding compound comprises alight chain constant region, for example a human light chain constantregion, such as lambda or kappa human light chain region or variantthereof.

In another embodiment, the invention relates to an isolated nucleicacid, for example DNA, encoding a binding compound of the presentinvention, for example an antibody (or binding fragment thereof) thatbinds to human IL-17A. In one embodiment, the isolated nucleic acidencodes a binding compound comprising at least one antibody light chainvariable (V_(L)) domain and at least one antibody heavy chain variable(V_(H)) domain, or binding fragments of these domains, wherein the V_(L)domain comprises at least a specified number of complementaritydetermining regions (CDRs) having a sequence selected from SEQ ID NOs:11-13, and the V_(H) domain comprises at least at least a specifiednumber of CDRs having a sequence selected from SEQ ID NOs:14-20, whereinthe specified number is one, two or three.

In another embodiment, the isolated nucleic acid encodes one or both ofthe light and heavy chain variable region sequences of SEQ ID NOs:5 and6, respectively. In yet another embodiment, the isolated nucleic acidencodes antibody 16C10 comprising a light chain having the sequence ofthe mature form of SEQ ID NO.: 2 and a heavy chain having the sequenceof the mature form of SEQ ID NO:4. In some embodiments, the isolatednucleic acid comprises nucleotides 58-717 of SEQ ID NO:1 or SEQ ID NO:62and in other embodiments the isolated nucleic acid comprises nucleotides58-1419 of SEQ ID NO:3 or SEQ ID NO:63. In yet another embodiment, theisolated nucleic acid comprises the sequence of SEQ ID NO:1 and thesequence of SEQ ID NO:3. In still yet another embodiment, the isolatednucleic acid comprises the sequence of SEQ ID NO:62 and the sequence ofSEQ ID NO:63. In some embodiments, the isolated nucleic acid encodesboth a light chain and a heavy chain on a single nucleic acid molecule,and in other embodiments the light and heavy chains are encoded on twoor more separate nucleic acid molecules.

In further embodiments, the present invention relates to expressionvectors comprising the isolated nucleic acids of the invention, whereinthe nucleic acid is operably linked to control sequences that arerecognized by a host cell when the host cell is transfected with thevector. In one embodiment, the expression vector has ATCC Accession No.PTA-7576 (hu16C10 in plasmid pAIL17AV1, deposited Jun. 28, 2006).

In another embodiment, the invention relates to a host cell comprisingan expression vector of the present invention. The invention furtherrelates to methods of producing a binding compound of the presentinvention comprising culturing a host cell harboring an expressionvector encoding the binding compound in culture medium, and isolatingthe binding compound from the host cell or culture medium.

The invention also relates to binding compounds, such as antibodies orbinding fragments thereof, that bind to the same epitope on human IL-17Aas antibodies 16C10, 4C3, 30C10, 12E6, 23E12 or 1D10; for example,antibodies that are able to cross-block binding of any of theseantibodies of the present invention, or antibodies that bind within theepitope defined by amino acid residues 74-85 of human IL-17A (SEQ IDNO.: 40).

The invention also relates to high affinity human IL-17A bindingcompounds, such as antibodies or binding fragments thereof, such asbinding compounds that bind with equilibrium dissociation constants(K_(d)) of 1000, 500, 100, 50, 20, 10, 5, 2 pM or less (i.e. higheraffinity). The invention also relates to binding compounds that havepotent biological activity, such as an IC₅₀ of 5000, 2000, 1000, 500 pMwhen measured in a biological activity assay where IL-17A stimulation iseffected at a concentration of 1000 pM (1 nM), such as IL-17A-stimulatedproduction of IL-6 from normal human dermal fibroblasts, foreskinfibroblasts, or synoviocytes. The invention also relates to bindingcompounds that have an IC₅₀ of 1000, 500, 200, 100, 50 pM or less whenmeasured in a biological activity assay where the IL-17A stimulation iseffected at a concentration of 100 pM, such as the Ba/F3-hIL-17Rc-mGCSFRproliferation assay. In general, the invention relates to bindingcompounds that are able to inhibit the activity of human IL-17A inbiological assays at concentrations that range from 10×, 5×, 2×, 1× andas low as 0.5× the concentration of IL-17A, when the concentration ofIL-17A is, e.g., 5, 10, 50, 100, 500 or 1000 pM or higher.

The invention also relates to binding compounds that are able to reduceIL-17A induced neutrophil recruitment to the lung by 50% or more whenadministered to mice to give a serum concentration of binding compoundof 50, 40, 30, 20 μg/ml or lower.

In one embodiment, the binding compound binds to cynomolgus monkeyIL-17A with an affinity (K_(d)) that is no more than 5, 10, or 20-foldlower than its affinity for human IL-17A. In another embodiment, thebinding compound binds to human IL-17A with an affinity (K_(d)) that is100, 500, 1000 or 2000-fold higher than its affinity for mouse or ratIL-17A.

The invention also relates to methods of treating subjects, includinghuman subjects, in need of treatment with the human IL-17A-bindingcompounds of the present invention. Such subjects may have aninflammatory or autoimmune disorder, such as rheumatoid arthritis,inflammatory bowel disease, psoriasis, multiple sclerosis, chronicobstructive pulmonary disease, cystic fibrosis, systemic scleroderma,allograft rejection, autoimmune myocarditis or peritoneal adhesions.Such methods of treatment may further comprise administering one or moreadditional therapeutic agents, such as immunosuppressive oranti-inflammatory agents. In one embodiment, the subject has beendiagnosed with an IL-17A-mediated disease. In another embodiment, thesubject has been diagnosed with rheumatoid arthritis. In yet anotherembodiment, the subject has been diagnosed with multiple sclerosis.

In a further embodiment, the invention provides methods of treatmentcomprising administration of a therapeutically effective amount of ananti-human IL-17A antibody or binding fragment in combination with oneor more other therapeutic agents. In one embodiment the othertherapeutic agent is an anti-human IL-23 antibody, or binding fragmentthereof. In various embodiments the anti-human IL-23 antibody orfragment is administered before, concurrently with, or after theanti-human IL-17A antibody or fragment. In one embodiment the antiIL-17A and anti IL-23 antibodies are administered together for a limitedtime during the acute phase of an adverse immunologic event, after whichtreatment with anti-IL-17A antibody is discontinued but treatment withanti IL-23 antibody is continued. In other embodiments, the one or moreother agent comprises an antagonist of IL-1β, IL-6 or TGF-β, for examplean anti-IL-6 or an anti-TGF-β antibody, or a combination of suchantagonists.

The invention also relates to compositions and formulations of thebinding compounds of the present invention, comprising the bindingcompound and a pharmaceutically acceptable carrier or diluent, andoptionally one or more immunosuppressive or anti-inflammatory agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows alignments of the light chain variable domains of severalanti-human IL-17A antibodies according to the present invention. Rat16C10 V_(L)=SEQ ID NO: 7; hum 16C10 V_(L)=SEQ ID NO: 5; rat 4C3V_(L)=SEQ ID NO: 21; hum 4C3 V_(L)=SEQ ID NO: 5; rat 23E12 V_(L)=SEQ IDNO: 45; rat 30C10 V_(L)=SEQ ID NO: 24; hum 30C10 V_(L)=SEQ ID NO: 22;rat 12E6 V_(L)=SEQ ID NO: 32; rat 1D10 V_(L)=SEQ ID NO: 54. CDRs areindicated (and are provided at Table 3). Numbering is according to Kabatet al. (1991) “Sequences of Proteins of Immunological Interest”, U.S.Department of Health and Human Services, NIH Pub. 91-3242, 5th Ed.,referred to herein as “Kabat et al. (1991)”.

FIG. 1B shows alignments of the heavy chain variable domains of severalanti-human IL-17A antibodies according to the present invention. Rat16C10 V_(H)=SEQ ID NO: 8; hum 16C10 V_(H)=SEQ ID NO: 6; rat 4C3V_(H)=SEQ ID NO: 8; hum 4C3 V_(H)=SEQ ID NO: 6; rat 23E12 V_(H)=SEQ IDNO: 47; rat 30C10 V_(H)=SEQ ID NO: 25; hum 30C10 V_(H)=SEQ ID NO: 23;rat 12E6 V_(H)=SEQ ID NO: 33; rat 1D10 V_(H)=SEQ ID NO: 55. CDRs areindicated (and are provided at Table 4). Numbering is according to Kabatet al. (1991).

FIG. 2A shows the amino acid sequence of the light chain of humanizedanti-IL-17A antibody 16C10 according to the present invention (themature form of SEQ ID NO: 2, i.e. residues 1-220). CDRs are indicated.

FIG. 2B shows the amino acid sequence for the heavy chain of humanizedanti-IL-17A antibody 16C10 according to the present invention (themature form of SEQ ID NO: 4, i.e. residues 1-454). CDRs are indicated.

FIGS. 3A-3D shows the effects of anti-IL-17A antibody treatments onpathology in the CIA mouse model of rheumatoid arthritis. Treatmentsinclude administration of anti-IL-17A antibody 1D10 (at 28, 7, and 2mg/kg) and administration of an isotype control (7 mg/kg).

FIG. 3A presents visual disease severity score (DSS), a measure ofvisual paw swelling and redness, as a function of antibody treatment.Scoring is: 0=paw appears the same as control (untreated) paw;1=inflammation of one finger on a given paw; 2=inflammation of twofingers or the palm of a given paw; 3=inflammation of the palm andfinger(s) of a given paw.

FIG. 3B presents cartilage damage (by histopathology) as a function ofantibody treatment. Scoring is: 0=normal; 1=minimal, 2=mild; 3=moderate;4=severe.

FIG. 3C presents bone erosion (by histopathology) as a function ofantibody treatment. Scoring is: 0=normal; 1=minimal, 2=mild; 3=moderate;4=severe.

FIG. 3D presents bone erosion (by histopathology) for paws from CIA micethat scored 2 or 3 in visual DSS, i.e. highly inflamed paws. rIgG1 is anisotype control antibody. Scoring is: 0=normal; 1=minimal, 2=mild;3=moderate; 4=severe.

FIG. 4 presents % BAL neutrophil (a measure of neutrophil recruitment tothe lung) in mice that had been treated intranasally with human IL-17A,as a function of the serum concentration of various anti-human IL-17Aantibodies of the present invention (1D10, 16C10, 4C3) and an isotypecontrol. Solid triangles represent control experiments without any addedanti-IL-17A antibody, and the leftmost data points (open triangles) areunstimulated controls.

FIG. 5A shows a nucleotide sequence (SEQ ID NO:62) encoding the lightchain of humanized anti-IL-17A antibody 16C10.

FIG. 5B shows a nucleotide sequence encoding the heavy chain ofhumanized anti-IL-17A antibody 16C10 (SEQ ID NO:63).

DETAILED DESCRIPTION I. Definitions

So that the invention may be more readily understood, certain technicaland scientific terms are specifically defined below. Unless specificallydefined elsewhere in this document, all other technical and scientificterms used herein have the meaning commonly understood by one ofordinary skill in the art to which this invention belongs.

As used herein, including the appended claims, the singular forms ofwords such as “a,” “an,” and “the,” include their corresponding pluralreferences unless the context clearly dictates otherwise.

“Activation,” “stimulation,” and “treatment,” as it applies to cells orto receptors, may have the same meaning, e.g., activation, stimulation,or treatment of a cell or receptor with a ligand, unless indicatedotherwise by the context or explicitly. “Ligand” encompasses natural andsynthetic ligands, e.g., cytokines, cytokine variants, analogues,muteins, and binding compounds derived from antibodies. “Ligand” alsoencompasses small molecules, e.g., peptide mimetics of cytokines andpeptide mimetics of antibodies. “Activation” can refer to cellactivation as regulated by internal mechanisms as well as by external orenvironmental factors. “Response,” e.g., of a cell, tissue, organ, ororganism, encompasses a change in biochemical or physiological behavior,e.g., concentration, density, adhesion, or migration within a biologicalcompartment, rate of gene expression, or state of differentiation, wherethe change is correlated with activation, stimulation, or treatment, orwith internal mechanisms such as genetic programming.

“Activity” of a molecule may describe or refer to the binding of themolecule to a ligand or to a receptor, to catalytic activity; to theability to stimulate gene expression or cell signaling, differentiation,or maturation; to antigenic activity, to the modulation of activities ofother molecules, and the like. “Activity” of a molecule may also referto activity in modulating or maintaining cell-to-cell interactions,e.g., adhesion, or activity in maintaining a structure of a cell, e.g.,cell membranes or cytoskeleton. “Activity” can also mean specificactivity, e.g., [catalytic activity]/[mg protein], or [immunologicalactivity]/[mg protein], concentration in a biological compartment, orthe like. “Activity” may refer to modulation of components of the innateor the adaptive immune systems. “Proliferative activity” encompasses anactivity that promotes, that is necessary for, or that is specificallyassociated with, e.g., normal cell division, as well as cancer, tumors,dysplasia, cell transformation, metastasis, and angiogenesis.

“Administration” and “treatment,” as it applies to an animal, human,experimental subject, cell, tissue, organ, or biological fluid, refersto contact of an exogenous pharmaceutical, therapeutic, diagnosticagent, or composition to the animal, human, subject, cell, tissue,organ, or biological fluid. “Administration” and “treatment” can refer,e.g., to therapeutic, pharmacokinetic, diagnostic, research, andexperimental methods. Treatment of a cell encompasses contact of areagent to the cell, as well as contact of a reagent to a fluid, wherethe fluid is in contact with the cell. “Administration” and “treatment”also means in vitro and ex vivo treatments, e.g., of a cell, by areagent, diagnostic, binding compound, or by another cell. “Treatment,”as it applies to a human, veterinary, or research subject, refers totherapeutic treatment, prophylactic or preventative measures, toresearch and diagnostic applications. “Treatment” as it applies to ahuman, veterinary, or research subject, or cell, tissue, or organ,encompasses contact of an IL-17A agonist or IL-17A antagonist to a humanor animal subject, a cell, tissue, physiological compartment, orphysiological fluid. “Treatment of a cell” also encompasses situationswhere the IL-17A agonist or IL-17A antagonist contacts IL-17A receptor,e.g., in the fluid phase or colloidal phase, but also situations wherethe agonist or antagonist does not contact the cell or the receptor.

“Treat” or “treating” means to administer a therapeutic agent, such as acomposition containing any of the binding compounds of the presentinvention, internally or externally to a patient having one or moredisease symptoms for which the agent has known therapeutic activity.Typically, the agent is administered in an amount effective to alleviateone or more disease symptoms in the treated patient or population,whether by inducing the regression of or inhibiting the progression ofsuch symptom(s) by any clinically measurable degree. The amount of atherapeutic agent that is effective to alleviate any particular diseasesymptom (also referred to as the “therapeutically effective amount”) mayvary according to factors such as the disease state, age, and weight ofthe patient, and the ability of the drug to elicit a desired response inthe patient. Whether a disease symptom has been alleviated can beassessed by any clinical measurement typically used by physicians orother skilled healthcare providers to assess the severity or progressionstatus of that symptom. While an embodiment of the present invention(e.g., a treatment method or article of manufacture) may not beeffective in alleviating the target disease symptom(s) in every patient,it should alleviate the target disease symptom(s) in a statisticallysignificant number of patients as determined by any statistical testknown in the art such as the Student's t-test, the chi²-test, the U-testaccording to Mann and Whitney, the Kruskal-Wallis test (H-test),Jonckheere-Terpstra-test and the Wilcoxon-test.

Four variants of human IL-17A protein are referred to herein. i) As usedherein, the terms “human IL-17A” and “native human IL-17A” (“huIL-17A”and “humIL-17A”) refer to the mature forms (i.e. residues 24-155) ofhuman IL-17A protein accession numbers NP_(—)002181 and AAT22064, andnaturally occurring variants and polymorphisms thereof ii) As usedherein, the term “rhIL-17A” refers to a recombinant derivative of nativehuman IL-17A in which two additional amino acids (LE) are appended atthe N-terminus of the mature form of native human IL-17A. Thisnomenclature is adopted for convenience in referring to various forms ofIL-17A, and may not match usage in the literature. iii) As used herein,the term “FLAG-huIL-17A” refers to a variant of native human IL-17Ahaving an N-terminal FLAG® peptide tag appended. In some experiments theFLAG-huIL-17A is biotinylated. iv) R&D Systems human IL-17A referred toherein is residues 20-155 of human IL-17A protein accession numbersNP_(—)002181 and AAT22064, with an additional N-terminal methionine.Table 1 is a summary of the variant N-termini of the IL-17A moleculesreferenced herein.

TABLE 1 Variant Forms of Human IL-17A SEQ ID IL-17A VariantSequence (N→C) NO.: huIL-17A (native)          GITIPRN . . . VHHVA 40rhIL-17A        LEGITIPRN . . . VHHVA 41 FLAG-huIL-17ADYKDDDDKLGITIPRN . . . VHHVA 42 R & D IL-17A    MIVKAGITIPRN . . . VHHVA 43

Unless otherwise noted, any IL-17A used in the experiments describedherein that is produced using adenoviral vectors is rhIL-17A. The term“IL-17A” refers to generally to human IL-17A, native or recombinant, andnon-human homologs of human IL-17A. Unless otherwise indicated, molarconcentrations of IL-17A are calculated using the molecular weight of ahomodimer of IL-17A (e.g., 30 kDa for human IL-17A).

As used herein, the term “antibody” refers to any form of antibody thatexhibits the desired biological activity. Thus, it is used in thebroadest sense and specifically covers, but is not limited to,monoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies). As used herein, the terms “IL-17A binding fragment or“binding fragment” of an antibody (the “parental antibody”) encompass afragment or a derivative of an antibody, typically including at least aportion of the antigen binding or variable regions (e.g. one or moreCDRs) of the parental antibody, that retains at least some of thebinding specificity of the parental antibody. Examples of antibodybinding fragments include, but are not limited to, Fab, Fab′, F(ab′)₂,and Fv fragments; diabodies; linear antibodies; single-chain antibodymolecules, e.g., sc-Fv; and multispecific antibodies formed fromantibody fragments. Typically, a binding fragment or derivative retainsat least 10% of its IL-17A binding activity when that activity isexpressed on a molar basis. Preferably, a binding fragment or derivativeretains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of theIL-17A binding affinity as the parental antibody. It is also intendedthat an IL-17A binding fragment can include conservative amino acidsubstitutions (referred to as “conservative variants” of the antibody)that do not substantially alter its biologic activity. The term “bindingcompound” refers to both antibodies and binding fragments thereof.

A “Fab fragment” is comprised of one light chain and the C_(H)1 andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule.

An “Fc” region contains two heavy chain fragments comprising the C_(H)1and C_(H)2 domains of an antibody. The two heavy chain fragments areheld together by two or more disulfide bonds and by hydrophobicinteractions of the CH3 domains.

A “Fab′ fragment” contains one light chain and a portion of one heavychain that contains the V_(H) domain and the C_(H)1 domain and also theregion between the C_(H)1 and C_(H)2 domains, such that an interchaindisulfide bond can be formed between the two heavy chains of two Fab′fragments to form a F(ab′)₂ molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H)1 and C_(H)² domains, such that an interchain disulfide bond is formed between thetwo heavy chains. A F(ab′)₂ fragment thus is composed of two Fab′fragments that are held together by a disulfide bond between the twoheavy chains.

The “Fv region” comprises the variable regions from both the heavy andlight chains, but lacks the constant regions.

The term “single-chain Fv” or “scFv” antibody refers to antibodyfragments comprising the V_(H) and V_(L) domains of an antibody, whereinthese domains are present in a single polypeptide chain. Generally, theFv polypeptide further comprises a polypeptide linker between the V_(H)and V_(L) domains which enables the scFv to form the desired structurefor antigen binding. For a review of scFv, see Pluckthun (1994) THEPHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Mooreeds. Springer-Verlag, New York, pp. 269-315. See also, InternationalPatent Application Publication No. WO 88/01649 and U.S. Pat. Nos.4,946,778 and 5,260,203.

A “domain antibody” is an immunologically functional immunoglobulinfragment containing only the variable region of a heavy chain or thevariable region of a light chain. In some instances, two or more V_(H)regions are covalently joined with a peptide linker to create a bivalentdomain antibody. The two V_(H) regions of a bivalent domain antibody maytarget the same or different antigens.

A “bivalent antibody” comprises two antigen binding sites. In someinstances, the two binding sites have the same antigen specificities.However, bivalent antibodies may be bispecific (see below).

As used herein, unless otherwise indicated, an “anti-IL-17A” antibodyrefers to an antibody that is raised against human IL-17A or a variantthereof, such as huIL-17A, rhIL-17A, FLAG-huIL-17A and R&D IL-17A, orany antigenic fragment thereof.

The term “monoclonal antibody”, as used herein, refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic epitope. In contrast, conventional(polyclonal) antibody preparations typically include a multitude ofantibodies directed against (or specific for) different epitopes. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler et al. (1975) Nature 256: 495, or maybe made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al. (1991)Nature 352: 624-628 and Marks et al. (1991), J. Mol. Biol. 222: 581-597,for example. See also Presta (2005) J. Allergy Clin. Immunol. 116:731.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855).

As used herein, a “chimeric antibody” is an antibody having the variabledomain from a first antibody and constant domain from a second antibody,where the first and second antibodies are from different species.Typically the variable domains are obtained from an antibody from anexperimental animal (the “parental antibody”), such as a rodent, and theconstant domain sequences are obtained from human antibodies, so thatthe resulting chimeric antibody will be less likely to elicit an adverseimmune response in a human subject than the parental rodent antibody.

The monoclonal antibodies herein also include camelized single domainantibodies. See, e.g., Muyldermans et al. (2001) Trends Biochem. Sci.26:230; Reichmann et al. (1999) J. Immunol. Methods 231:25; WO 94/04678;WO 94/25591; U.S. Pat. No. 6,005,079, which are hereby incorporated byreference in their entireties). In one embodiment, the present inventionprovides single domain antibodies comprising two V_(H) domains withmodifications such that single domain antibodies are formed.

As used herein, the term “diabodies” refers to small antibody fragmentswith two antigen-binding sites, which fragments comprise a heavy chainvariable domain (V_(H)) connected to a light chain variable domain(V_(L)) in the same polypeptide chain (V_(H)-V_(L) or V_(L)-V_(H)). Byusing a linker that is too short to allow pairing between the twodomains on the same chain, the domains are forced to pair with thecomplementary domains of another chain and create two antigen-bindingsites. Diabodies are described more fully in, e.g., EP 404,097; WO93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. For a review of engineered antibody variants generally seeHolliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

As used herein, the term “humanized antibody” refers to forms ofantibodies that contain sequences from both human and non-human (e.g.,murine, rat) antibodies. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the hypervariable loopscorrespond to those of a non-human immunoglobulin, and all orsubstantially all of the framework (FR) regions are those of a humanimmunoglobulin sequence. The humanized antibody may optionally compriseat least a portion of a human immunoglobulin constant region (Fc).

The antibodies of the present invention also include antibodies withmodified (or blocked) Fc regions to provide altered effector functions.See, e.g., U.S. Pat. No. 5,624,821; WO2003/086310; WO2005/120571;WO2006/0057702. Such modification can be used to enhance or suppressvarious reactions of the immune system, with possible beneficial effectsin diagnosis and therapy. Alterations of the Fc region include aminoacid changes (substitutions, deletions and insertions), glycosylation ordeglycosylation, and adding multiple Fc. Changes to the Fc can alsoalter the half-life of antibodies in therapeutic antibodies, enablingless frequent dosing and thus increased convenience and decreased use ofmaterial. See Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734-35.

The term “fully human antibody” refers to an antibody that compriseshuman immunoglobulin protein sequences only. A fully human antibody maycontain murine carbohydrate chains if produced in a mouse, in a mousecell, or in a hybridoma derived from a mouse cell. Similarly, “mouseantibody” refers to an antibody that comprises mouse immunoglobulinsequences only. Alternatively, a fully human antibody may contain ratcarbohydrate chains if produced in a rat, in a rat cell, or in ahybridoma derived from a rat cell. Similarly, “rat antibody” refers toan antibody that comprises rat immunoglobulin sequences only.

As used herein, the term “hypervariable region” refers to the amino acidresidues of an antibody which are responsible for antigen-binding. Thehypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e. residues 24-34(CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3) in the light chain variabledomain and residues 31-35 (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) inthe heavy chain variable domain; Kabat et al. (1991) Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md.) and/or those residues froma “hypervariable loop” (i.e. residues 26-32 (CDRL1), 50-52 (CDRL2) and91-96 (CDRL3) in the light chain variable domain and 26-32 (CDRH1),53-55 (CDRH2) and 96-101 (CDRH3) in the heavy chain variable domain;Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917). As used herein, theterm “framework” or “FR” residues refers to those variable domainresidues other than the hypervariable region residues defined herein asCDR residues.

“Binding substance” refers to a molecule, small molecule, macromolecule,antibody, a fragment or analogue thereof, or soluble receptor, capableof binding to a target. “Binding substance” also may refer to a complexof molecules, e.g., a non-covalent complex, to an ionized molecule, andto a covalently or non-covalently modified molecule, e.g., modified byphosphorylation, acylation, cross-linking, cyclization, or limitedcleavage, that is capable of binding to a target. “Binding substance”may also refer to a molecule capable of binding to a target incombination with a stabilizer, excipient, salt, buffer, solvent, oradditive. “Binding” may be defined as an association of the bindingsubstance with a target where the association results in reduction inthe normal Brownian motion of the binding substance, in cases where thebinding substance can be dissolved or suspended in solution.

“Conservatively modified variants” or “conservative substitution” refersto substitutions of amino acids in a protein with other amino acidshaving similar characteristics (e.g. charge, side-chain size,hydrophobicity/hydrophilicity, backbone conformation and rigidity,etc.), such that the changes can frequently be made without altering thebiological activity of the protein. Those of skill in this art recognizethat, in general, single amino acid substitutions in non-essentialregions of a polypeptide do not substantially alter biological activity(see, e.g., Watson et al. (1987) Molecular Biology of the Gene, TheBenjamin/Cummings Pub. Co., p. 224 (4th Ed.)). In addition,substitutions of structurally or functionally similar amino acids areless likely to disrupt biological activity. Various embodiments of thebinding compounds of the present invention comprise polypeptide chainswith sequences that include up to 0 (no changes), 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 12, 15, 20 or more conservative amino acid substitutions whencompared with the specific amino acid sequences disclosed herein, e.g.SEQ ID NOs: 2, 4, 5, or 6. As used herein, the phrase “up to X”conservative amino acid substitutions includes 0 substitutions and anynumber of substitutions up to and including X substitutions. Suchexemplary substitutions are preferably made in accordance with those setforth in Table 2 as follows:

TABLE 2 Exemplary Conservative Amino Acid Substitutions Original residueConservative substitution Ala (A) Gly; Ser Arg (R) Lys; His Asn (N) Gln;His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly(G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg;His Met (M) Leu; Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) ThrThr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu

The terms “consists essentially of,” or variations such as “consistessentially of” or “consisting essentially of,” as used throughout thespecification and claims, indicate the inclusion of any recited elementsor group of elements, and the optional inclusion of other elements, ofsimilar or different nature than the recited elements, which do notmaterially change the basic or novel properties of the specified dosageregimen, method, or composition. As a nonlimiting example, a bindingcompound which consists essentially of a recited amino acid sequence mayalso include one or more amino acids that do not materially affect theproperties of the binding compound.

“Effective amount” encompasses an amount sufficient to ameliorate orprevent a symptom or sign of the medical condition. Effective amountalso means an amount sufficient to allow or facilitate diagnosis. Aneffective amount for a particular patient or veterinary subject may varydepending on factors such as the condition being treated, the overallhealth of the patient, the method route and dose of administration andthe severity of side affects (see, e.g., U.S. Pat. No. 5,888,530 issuedto Netti, et al.). An effective amount can be the maximal dose or dosingprotocol that avoids significant side effects or toxic effects. Theeffect will result in an improvement of a diagnostic measure orparameter by at least 5%, usually by at least 10%, more usually at least20%, most usually at least 30%, preferably at least 40%, more preferablyat least 50%, most preferably at least 60%, ideally at least 70%, moreideally at least 80%, and most ideally at least 90%, where 100% isdefined as the diagnostic parameter shown by a normal subject (see,e.g., Maynard, et al. (1996) A Handbook of SOPs for Good ClinicalPractice, Interpharm Press, Boca Raton, Fla.; Dent (2001) GoodLaboratory and Good Clinical Practice, Urch Publ., London, UK).

“Exogenous” refers to substances that are produced outside an organism,cell, or human body, depending on the context. “Endogenous” refers tosubstances that are produced within a cell, organism, or human body,depending on the context.

“Homology” refers to sequence similarity between two polynucleotidesequences or between two polypeptide sequences. When a position in bothof the two compared sequences is occupied by the same base or amino acidmonomer subunit, e.g., if a position in each of two DNA molecules isoccupied by adenine, then the molecules are homologous at that position.The percent of homology between two sequences is a function of thenumber of matching or homologous positions shared by the two sequencesdivided by the number of positions compared ×100. For example, if 6 of10 of the positions in two sequences are matched or homologous when thesequences are optimally aligned then the two sequences are 60%homologous. Generally, the comparison is made when two sequences arealigned to give maximum percent homology.

“Immune condition” or “immune disorder” encompasses, e.g., pathologicalinflammation, an inflammatory disorder, and an autoimmune disorder ordisease. “Immune condition” also refers to infections, persistentinfections, and proliferative conditions, such as cancer, tumors, andangiogenesis, including infections, tumors, and cancers that resisteradication by the immune system. “Cancerous condition” includes, e.g.,cancer, cancer cells, tumors, angiogenesis, and precancerous conditionssuch as dysplasia.

“Inflammatory disorder” means a disorder or pathological condition wherethe pathology results, in whole or in part, from, e.g., a change innumber, change in rate of migration, or change in activation, of cellsof the immune system. Cells of the immune system include, e.g., T cells,B cells, monocytes or macrophages, antigen presenting cells (APCs),dendritic cells, microglia, NK cells, NKT cells, neutrophils,eosinophils, mast cells, or any other cell specifically associated withthe immunology, for example, cytokine-producing endothelial orepithelial cells.

“Isolated binding compound” refers to the purification status of abinding compound and in such context means the molecule is substantiallyfree of other biological molecules such as nucleic acids, proteins,lipids, carbohydrates, or other material such as cellular debris andgrowth media. Generally, the term “isolated” is not intended to refer toa complete absence of such material or to an absence of water, buffers,or salts, unless they are present in amounts that substantiallyinterfere with experimental or therapeutic use of the binding compoundas described herein.

“Isolated nucleic acid molecule” means a DNA or RNA of genomic, mRNA,cDNA, or synthetic origin or some combination thereof which is notassociated with all or a portion of a polynucleotide in which theisolated polynucleotide is found in nature, or is linked to apolynucleotide to which it is not linked in nature. For purposes of thisdisclosure, it should be understood that “a nucleic acid moleculecomprising” a particular nucleotide sequence does not encompass intactchromosomes. Isolated nucleic acid molecules “comprising” specifiednucleic acid sequences may include, in addition to the specifiedsequences, coding sequences for up to ten or even up to twenty or moreother proteins or portions thereof, or may include operably linkedregulatory sequences that control expression of the coding region of therecited nucleic acid sequences, and/or may include vector sequences.

The phrase “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to use promoters,polyadenylation signals, and enhancers.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

As used herein, “polymerase chain reaction” or “PCR” refers to aprocedure or technique in which minute amounts of a specific piece ofnucleic acid, RNA and/or DNA, are amplified as described in, e.g., U.S.Pat. No. 4,683,195. Generally, sequence information from the ends of theregion of interest or beyond needs to be available, such thatoligonucleotide primers can be designed; these primers will be identicalor similar in sequence to opposite strands of the template to beamplified. The 5′ terminal nucleotides of the two primers can coincidewith the ends of the amplified material. PCR can be used to amplifyspecific RNA sequences, specific DNA sequences from total genomic DNA,and cDNA transcribed from total cellular RNA, bacteriophage or plasmidsequences, etc. See generally Mullis et al. (1987) Cold Spring HarborSymp. Quant. Biol. 51:263; Erlich, ed., (1989) PCR TECHNOLOGY (StocktonPress, N.Y.) As used herein, PCR is considered to be one, but not theonly, example of a nucleic acid polymerase reaction method foramplifying a nucleic acid test sample comprising the use of a knownnucleic acid as a primer and a nucleic acid polymerase to amplify orgenerate a specific piece of nucleic acid.

As used herein, the term “germline sequence” refers to a sequence ofunrearranged immunoglobulin DNA sequences. Any suitable source ofunrearranged immunoglobulin may be used.

“Inhibitors” and “antagonists,” or “activators” and “agonists,” refer toinhibitory or activating molecules, respectively, e.g., for theactivation of, e.g., a ligand, receptor, cofactor, a gene, cell, tissue,or organ. A modulator of, e.g., a gene, a receptor, a ligand, or a cell,is a molecule that alters an activity of the gene, receptor, ligand, orcell, where activity can be activated, inhibited, or altered in itsregulatory properties. The modulator may act alone, or it may use acofactor, e.g., a protein, metal ion, or small molecule. Inhibitors arecompounds that decrease, block, prevent, delay activation, inactivate,desensitize, or down regulate, e.g., a gene, protein, ligand, receptor,or cell. Activators are compounds that increase, activate, facilitate,enhance activation, sensitize, or up regulate, e.g., a gene, protein,ligand, receptor, or cell. An inhibitor may also be defined as acompound that reduces, blocks, or inactivates a constitutive activity.An “agonist” is a compound that interacts with a target to cause orpromote an increase in the activation of the target. An “antagonist” isa compound that opposes the actions of an agonist. An antagonistprevents, reduces, inhibits, or neutralizes the activity of an agonist.An antagonist can also prevent, inhibit, or reduce constitutive activityof a target, e.g., a target receptor, even where there is no identifiedagonist.

To examine the extent of inhibition, for example, samples or assayscomprising a given, e.g., protein, gene, cell, or organism, are treatedwith a potential activator or inhibitor and are compared to controlsamples without the inhibitor. Control samples, i.e., samples nottreated with antagonist, are assigned a relative activity value of 100%Inhibition is achieved when the activity value relative to the controlis about 90% or less, typically 85% or less, more typically 80% or less,most typically 75% or less, generally 70% or less, more generally 65% orless, most generally 60% or less, typically 55% or less, usually 50% orless, more usually 45% or less, most usually 40% or less, preferably 35%or less, more preferably 30% or less, still more preferably 25% or less,and most preferably less than 25%. Activation is achieved when theactivity value relative to the control is about 110%, generally at least120%, more generally at least 140%, more generally at least 160%, oftenat least 180%, more often at least 2-fold, most often at least 2.5-fold,usually at least 5-fold, more usually at least 10-fold, preferably atleast 20-fold, more preferably at least 40-fold, and most preferablyover 40-fold higher.

Endpoints in activation or inhibition can be monitored as follows.Activation, inhibition, and response to treatment, e.g., of a cell,physiological fluid, tissue, organ, and animal or human subject, can bemonitored by an endpoint. The endpoint may comprise a predeterminedquantity or percentage of, e.g., indicia of inflammation, oncogenicity,or cell degranulation or secretion, such as the release of a cytokine,toxic oxygen, or a protease. The endpoint may comprise, e.g., apredetermined quantity of ion flux or transport; cell migration; celladhesion; cell proliferation; potential for metastasis; celldifferentiation; and change in phenotype, e.g., change in expression ofgene relating to inflammation, apoptosis, transformation, cell cycle, ormetastasis (see, e.g., Knight (2000) Ann. Clin. Lab. Sci. 30:145-158;Hood and Cheresh (2002) Nature Rev. Cancer 2:91-100; Timme, et al.(2003) Curr. Drug Targets 4:251-261; Robbins and Itzkowitz (2002) Med.Clin. North Am. 86:1467-1495; Grady and Markowitz (2002) Annu. Rev.Genomics Hum. Genet. 3:101-128; Bauer, et al. (2001) Glia 36:235-243;Stanimirovic and Satoh (2000) Brain Pathol. 10:113-126).

An endpoint of inhibition is generally 75% of the control or less,preferably 50% of the control or less, more preferably 25% of thecontrol or less, and most preferably 10% of the control or less.Generally, an endpoint of activation is at least 150% the control,preferably at least two times the control, more preferably at least fourtimes the control, and most preferably at least ten times the control.

“Ligand” refers, e.g., to a small molecule, peptide, polypeptide, andmembrane associated or membrane-bound molecule, or complex thereof, thatcan act as an agonist or antagonist of a receptor. “Ligand” alsoencompasses an agent that is not an agonist or antagonist, but that canbind to the receptor. Moreover, “ligand” includes a membrane-boundligand that has been changed, e.g., by chemical or recombinant methods,to a soluble version of the membrane-bound ligand. By convention, wherea ligand is membrane-bound on a first cell, the receptor usually occurson a second cell. The second cell may have the same or a differentidentity as the first cell. A ligand or receptor may be entirelyintracellular, that is, it may reside in the cytosol, nucleus, or someother intracellular compartment. The ligand or receptor may change itslocation, e.g., from an intracellular compartment to the outer face ofthe plasma membrane. The complex of a ligand and receptor is termed a“ligand receptor complex.” Where a ligand and receptor are involved in asignaling pathway, the ligand occurs at an upstream position and thereceptor occurs at a downstream position of the signaling pathway.

“Small molecule” is defined as a molecule with a molecular weight thatis less than 10 kDa, typically less than 2 kDa, preferably less than 1kDa, and most preferably less than about 500 Da. Small moleculesinclude, but are not limited to, inorganic molecules, organic molecules,organic molecules containing an inorganic component, moleculescomprising a radioactive atom, synthetic molecules, peptide mimetics,and antibody mimetics. As a therapeutic, a small molecule may be morepermeable to cells, less susceptible to degradation, and less apt toelicit an immune response than large molecules. Small molecules, such aspeptide mimetics of antibodies and cytokines, as well as small moleculetoxins, have been described (see, e.g., Casset, et al. (2003) Biochem.Biophys. Res. Commun. 307:198-205; Muyldermans (2001) J. Biotechnol.74:277-302; Li (2000) Nat. Biotechnol. 18:1251-1256; Apostolopoulos, etal. (2002) Curr. Med. Chem. 9:411-420; Monfardini, et al. (2002) Curr.Pharm. Des. 8:2185-2199; Domingues, et al. (1999) Nat. Struct. Biol.6:652-656; Sato and Sone (2003) Biochem. J. 371:603-608; U.S. Pat. No.6,326,482 issued to Stewart, et al).

“Specifically” or “selectively” binds, when referring to aligand/receptor, antibody/antigen, or other binding pair, indicates abinding reaction that is determinative of the presence of the protein ina heterogeneous population of proteins and other biologics. Thus, underdesignated conditions, a specified ligand binds to a particular receptorand does not bind in a significant amount to other proteins present inthe sample. The antibody, or binding compound derived from theantigen-binding site of an antibody, of the contemplated method binds toits antigen, or a variant or mutein thereof, with an affinity that is atleast two fold greater, preferably at least ten times greater, morepreferably at least 20-times greater, and most preferably at least100-times greater than the affinity with any other antigen. In apreferred embodiment the antibody will have an affinity that is greaterthan about 10⁹ M⁻¹, as determined, e.g., by Scatchard analysis (Munsenet al. (1980) Analyt. Biochem. 107:220-239).

As used herein, the term “immunomodulatory agent” refers to natural orsynthetic agents that suppress or modulate an immune response. Theimmune response can be a humoral or cellular response. Immunomodulatoryagents encompass immunosuppressive or anti-inflammatory agents.

“Immunosuppressive agents”, “immunosuppressive drugs”, or“immunosuppressants” as used herein are therapeutics that are used inimmunosuppressive therapy to inhibit or prevent activity of the immunesystem. Clinically they are used to prevent the rejection oftransplanted organs and tissues (e.g. bone marrow, heart, kidney,liver), and/or in the treatment of autoimmune diseases or diseases thatare most likely of autoimmune origin (e.g. rheumatoid arthritis,myasthenia gravis, systemic lupus erythematosus, ulcerative colitis,multiple sclerosis) Immunosuppressive drugs can be classified as:glucocorticoids; cytostatics; antibodies (biological responsemodifiers); drugs acting on immunophilins; other drugs, including knownchemotherpeutic agents used in the treatment of proliferative disorders.For multiple sclerosis, in particular, the antibodies of the presentinvention can be administered in conjunction with a new class of myelinbinding protein-like therapeutics, known as copaxones.

“Anti-inflammatory agents” or “anti-inflammatory drugs” refer to bothsteroidal and non-steroidal therapeutics. Steroids, also known ascorticosteroids, are drugs that closely resemble cortisol, a hormoneproduced naturally by adrenal glands. Steroids are used as the maintreatment for certain inflammatory conditions, such as: systemicvasculitis (inflammation of blood vessels); and myositis (inflammationof muscle). Steroids might also be used selectively to treatinflammatory conditions such as: rheumatoid arthritis (chronicinflammatory arthritis occurring in joints on both sides of the body);systemic lupus erythematosus (a generalized disease caused by abnormalimmune system function); Sjögren's syndrome (chronic disorder thatcauses dry eyes and a dry mouth).

Non-steroidal anti-inflammatory drugs, usually abbreviated to NSAIDs,are drugs with analgesic, antipyretic and anti-inflammatory effects—theyreduce pain, fever and inflammation. The term “non-steroidal” is used todistinguish these drugs from steroids, which (amongst a broad range ofother effects) have a similar eicosanoid-depressing, anti-inflammatoryaction. NSAIDs are generally indicated for the symptomatic relief of thefollowing conditions: rheumatoid arthritis; osteoarthritis; inflammatoryarthropathies (e.g. ankylosing spondylitis, psoriatic arthritis,Reiter's syndrome); acute gout; dysmenorrhoea; metastatic bone pain;headache and migraine; postoperative pain; mild-to-moderate pain due toinflammation and tissue injury; pyrexia; and renal colic. NSAIDs includesalicylates, arlyalknoic acids, 2-arylpropionic acids (profens),N-arylanthranilic acids (fenamic acids), oxicams, coxibs, andsulphonanilides.

Disease-modifying anti-rheumatic drugs (DMARDs) may be administered,often in combination with NSAIDs. Commonly prescribed DMARDs includehydroxychloroquine/chloroquine, methotrexate, gold therapy,sulfasalazine, and azathioprine.

II. Antibodies Specific for Human IL-17A

The present invention provides engineered anti-IL-17A antibodies anduses thereof to treat various inflammatory, immune and proliferativedisorders, including rheumatoid arthritis (RA), osteoarthritis,rheumatoid arthritis osteoporosis, inflammatory fibrosis (e.g.,scleroderma, lung fibrosis, and cirrhosis), inflammatory bowel disorders(e.g., Crohn's disease, ulcerative colitis and inflammatory boweldisease), asthma (including allergic asthma), allergies, COPD, multiplesclerosis, psoriasis and cancer.

Any suitable method for generating monoclonal antibodies may be used togenerate the anti-IL-17A antibodies of the present invention. Forexample, a recipient animal may be immunized with a linked or unlinked(e.g. naturally occurring) form of the IL-17A homodimer, or a fragmentthereof. Any suitable method of immunization can be used. Such methodscan include adjuvants, other immunostimulants, repeated boosterimmunizations, and the use of one or more immunization routes.

Any suitable form of IL-17A can be used as the immunogen (antigen) forthe generation of the non-human antibody specific for IL-17A, whichantibody can be screened for biological activity. The elicitingimmunogen may be full-length mature human IL-17A, including linked andnaturally occurring homodimers, or peptides thereof encompassing singleepitopes or multiple epitopes. The immunogen may be used alone or incombination with one or more immunogenicity enhancing agents known inthe art. The immunogen may be purified from a natural source or producedin a genetically modified cell. DNA encoding the immunogen may begenomic or non-genomic (e.g., cDNA) in origin. Immunogen-encoding DNAmay be expressed using suitable genetic vectors, including but notlimited to adenoviral vectors, adenoassociated viral vectors,baculoviral vectors, plasmids, and non-viral vectors, such as cationiclipids.

Any suitable method can be used to elicit an antibody response with thedesired biologic properties, e.g. to inhibit IL-17A binding to itsreceptor. In some embodiments, antibodies are raised in mammalian hostssuch as mice, rodents, primates, humans, etc. Techniques for preparingmonoclonal antibodies may be found in, e.g., Stites et al. (eds.) BASICAND CLINICAL IMMUNOLOGY (4th ed.) Lange Medical Publications, Los Altos,Calif., and references cited therein; Harlow and Lane (1988) ANTIBODIES:A LABORATORY MANUAL CSH Press; Goding (1986) MONOCLONAL ANTIBODIES:PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York, N.Y. Thus,monoclonal antibodies may be obtained by a variety of techniquesfamiliar to researchers skilled in the art. Typically, spleen cells froman animal immunized with a desired antigen are immortalized, commonly byfusion with a myeloma cell. See Kohler and Milstein (1976) Eur. J.Immunol. 6:511-519. Alternative methods of immortalization includetransformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods known in the art. See, e.g., Doyle et al. (eds.) (1994 andperiodic supplements) CELL AND TISSUE CULTURE: LABORATORY PROCEDURES,John Wiley and Sons, New York, N.Y. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen. The yield ofmonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host.

Other suitable techniques involve selection of libraries of antibodiesin phage or similar vectors. See, e.g., Huse et al., Science246:1275-1281 (1989); and Ward et al., Nature 341:544-546 (1989). Theantibodies of the present invention may be used without modification,e.g. as the parental rodent antibody, or with modifications tofacilitate their use as therapeutic agents in human subjects, such aschimeric or humanized antibodies. In some embodiments, the antibodieswill be labeled, covalently or non-covalently, with a substance thatprovides a detectable signal. A wide variety of labels and conjugationtechniques are known and are reported extensively in both the scientificand patent literature. Suitable labels include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent moieties,chemiluminescent moieties, magnetic particles, and the like. Patentsteaching the use of such labels include U.S. Pat. Nos. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.Also, recombinant immunoglobulins may be produced, see Cabilly U.S. Pat.No. 4,816,567; and Queen et al. (1989) Proc. Nat'l Acad. Sci. USA86:10029-10033; or made in transgenic mice, see Mendez et al. (1997)Nature Genetics 15:146-156; also see Abgenix and Medarex technologies.

Antibodies against predetermined fragments of IL-17A can be raised byimmunization of animals with conjugates of the predetermined fragment ofIL-17A with carrier proteins. Monoclonal antibodies are prepared fromcells secreting the desired antibody. These antibodies can be screenedfor binding to normal or defective IL-17A. These monoclonal antibodieswill usually bind with at least a K_(d) of about 1 μM, more usually atleast about 300, 30, 10, or 3 nM, preferably at least about 300, 100,30, 10, 3, or 1 pM. Because of the inverse relationship of K_(d) valuesand affinity, references to binding with a given K_(d) “or less” refersto binding with an affinity that is at least as high as the recitednumerical value, i.e. with a K_(d) that is at least as low as the citedvalue. Binding affinities may be determined by ELISA (see Examples 5-6,infra), or by Biacore® surface plasmon resonance spectroscopy, KinExA orECL methods (see Example 7, infra). Suitable non-human antibodies mayalso be identified using the biological assays described in Examples8-11 and 16-17, infra.

An exemplary method of producing anti-human IL-17A antibodies of thepresent invention is described at Example 2.

III. Humanization of IL-17A Specific Antibodies

Any suitable non-human antibody can be used as a source for thehypervariable region of an anti-IL-17A antibody of the presentinvention. Sources for non-human antibodies include, but are not limitedto, rodents (e.g. mouse, rat), Lagomorphs (including rabbits), cows, andnonhuman primates. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity and affinity.Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody, such asmodifications made to further refine antibody performance of the desiredbiological activity. For further details, see Jones et al. (1986) Nature321: 522-525; Reichmann et al. (1988) Nature 332: 323-329; and Presta(1992) Curr. Op. Struct. Biol. 2: 593-596.

Methods for recombinantly engineering and producing antibodies have beendescribed, e.g., by Boss et al. (U.S. Pat. No. 4,816,397), Cabilly etal. (U.S. Pat. No. 4,816,567), Law et al. (European Patent ApplicationPublication No. 438 310) and Winter (European Patent ApplicationPublication No. 239 400).

Amino acid sequence variants of humanized anti-IL-17A antibodies of thepresent invention may be prepared by introducing appropriate nucleotidechanges into the humanized anti-IL-17A antibody DNA, or by peptidesynthesis. Any combination of deletion, insertion, and substitution maybe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics. The amino acid changesalso may alter post-translational processing of the humanizedanti-IL-17A antibody, such as changing the number or position ofglycosylation sites.

One useful method for identifying residues or regions of a humanizedanti-IL-17A antibody that are preferred locations for mutagenesis iscalled “alanine scanning mutagenesis.” Cunningham and Wells (1989)Science 244: 1081-1085. A group of target residues is identified (e.g.,charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by aneutral or negatively charged amino acid (most preferably alanine orpolyalanine) to alter the interaction of the amino acids with IL-17A.The residues showing functional sensitivity to alanine substitutions arethen refined by introducing further amino acid substitutions. In oneembodiment, the effect of mutations at a given target codon isdetermined by alanine scanning or random mutagenesis followed byactivity and binding analysis of the resulting humanized anti-IL-17Aantibody variants.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includehumanized anti-IL-17A antibody with an N-terminal methionyl residue orthe antibody fused to an epitope tag. Other variants include the fusionof an enzyme or a polypeptide that increases the serum half-life of anantibody to the N- or C-terminus.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the humanizedanti-IL-17A antibody molecule removed and a different residue insertedin its place. The sites of greatest interest for substitutionalmutagenesis include the hypervariable loops, but FR alterations are alsocontemplated. Hypervariable region residues or FR residues involved inantigen binding are generally substituted in a relatively conservativemanner.

Other amino acid variants of the antibody alter the originalglycosylation pattern of the antibody, e.g. by eliminating one or morecarbohydrate moieties and/or adding one or more glycosylation sites.Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain. Thepresence of either of these tripeptide sequences in a polypeptidecreates a potential glycosylation site. O-linked glycosylation involvesattachment of N-acetylgalactosamine, galactose, or xylose to ahydroxyamino acid, most commonly serine or threonine, although5-hydroxyproline or 5-hydroxylysine may also be used.

Glycosylation sites can be added to the antibodies of the presentinvention by inserting one or more of the above-described tripeptidesequences (for N-linked glycosylation sites), or addition of one or moreserine or threonine residues (for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants ofhumanized IL-17A-specific antibody are prepared by a variety of methodsknown in the art. These methods include, but are not limited to,isolation from a natural source (in the case of naturally occurringamino acid sequence variants), or by oligonucleotide-mediated (orsite-directed) mutagenesis, PCR mutagenesis, or cassette mutagenesis.

Ordinarily, amino acid sequence variants of the humanized anti-IL-17Aantibody will have an amino acid sequence having at least 50% amino acidsequence identity with the original humanized antibody amino acidsequences of either the heavy or the light chain, preferably at least70%, 80%, 85%, 90%, and most preferably at least 95%. Identity orhomology with respect to this sequence is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical with the humanized anti-IL-17A residues when the sequences areoptimally aligned (i.e. after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity),and not considering any conservative substitutions as part of thesequence identity. None of N-terminal, C-terminal, or internalextensions, deletions, or insertions into the antibody sequence isconsidered to affect sequence identity or homology.

The humanized antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA, and IgE. In oneembodiment, the antibody is an IgG antibody. Any isotype of IgG can beused, including IgG₁, IgG₂, IgG₃, and IgG₄. Variants of the IgG isotypesare also contemplated. The humanized antibody may comprise sequencesfrom more than one class or isotype. Optimization of the necessaryconstant domain sequences to generate the desired biologic activity isreadily achieved by screening the antibodies in the biological assaysdescribed below in the Examples.

Likewise, either class of light chain can be used in the compounds andmethods herein. Specifically, kappa, lambda, or variants thereof areuseful in the present compounds and methods.

Any suitable portion of the CDR sequences from the non-human antibodycan be used to create the humanized antibodies of the present invention.The CDR sequences may be mutagenized by substitution, insertion ordeletion, although such mutations would be minimal because of the needto maintain IL-17A binding affinity and specificity. Typically, at least75% of the humanized antibody CDR residues will correspond to those ofthe non-human CDR residues, more often 90%, and most preferably greaterthan 95%, and frequently 100%.

Any suitable portion of the FR sequences from the human antibody can beused. The FR sequences can be mutagenized by substitution, insertion ordeletion of at least one residue such that the FR sequence is distinctfrom the human and non-human antibody sequence employed. It iscontemplated that such mutations would be minimal. Typically, at least75% of the humanized antibody residues will correspond to those of thehuman FR residues, more often 90%, and most preferably greater than 95%.

Also contemplated are chimeric antibodies or fragments thereof, so longas they exhibit the desired biological activity (U.S. Pat. No.4,816,567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855). As noted above, typical chimeric antibodies compriseconstant domain sequences from antibodies from one species linked to thevariable domain of an antigen-specific antibody obtained from adifferent species.

The binding compounds of the invention may comprise bispecificantibodies. As used herein, the term “bispecific antibody” refers to anantibody, typically a monoclonal antibody, having binding specificitiesfor at least two different antigenic epitopes. In one embodiment, theepitopes are from the same antigen. In another embodiment, the epitopesare from two different antigens. Methods for making bispecificantibodies are known in the art. For example, bispecific antibodies canbe produced recombinantly using the co-expression of two immunoglobulinheavy chain/light chain pairs. See, e.g., Milstein et al. (1983) Nature305: 537-39. Alternatively, bispecific antibodies can be prepared usingchemical linkage. See, e.g., Brennan, et al. (1985) Science 229: 81.Bispecific antibodies include bispecific antibody fragments. See, e.g.,Hollinger, et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90: 6444-48,Gruber, et al., J. Immunol. 152: 5368 (1994).

An exemplary method of humanizing anti-human IL-17A antibodies of thepresent invention is described at Example 3.

IV. Characterization of IL-17A Specific Antibodies

The methods described herein were used to generate monoclonal antibodiesimmunoreactive with human IL-17A, as described in greater detail inExamples 2 and 3. FIGS. 1A and 1B show sequence alignments of thevariable regions of the light and heavy chains, respectively, of variousanti-IL-17A antibodies of the present invention. CDR regions areindicated, and numbering is according to Kabat et al. (1991).

A plasmid containing the nucleic acid sequences encoding the humanized16C10 light and heavy chains was deposited pursuant to the BudapestTreaty on Jul. 28, 2006, with American Type Culture Collection(ATCC—Manassas, Va., USA) under Accession Number PTA-7675. Hybridomasexpressing antibodies 30C10 and 23E12 were deposited as JL7-30C10.C3 andJL7-23E12.B10, respectively, pursuant to the Budapest Treaty on Jul. 20,2006, with American Type Culture Collection (ATCC—Manassas, Va., USA)under Accession Numbers PTA-7739 and PTA-7740.

The light and heavy chain CDRs of various humanized antibodies of thepresent invention are provided at Tables 3 and 4, respectively. Inaddition, Table 4 provides additional CDRs for V_(H) of hu16C10 withvariable positions at which more than one amino acid can be used.

TABLE 3 Variable Light Chain CDR Sequences Antibody CDRL1 CDRL2 CDRL3hum 16C10 KSSQSLLFSENQKNYLA WTSTRQS QQSYYTPYT (SEQ ID NO: 11)(SEQ ID NO: 12) (SEQ ID NO: 13) hum 4C3 KSSQSLLFSENQKNYLA WTSTRQSQQSYYTPYT (SEQ ID NO: 11) (SEQ ID NO: 12) (SEQ ID NO: 13) hum 23E12QASEDIYSGLA GASRLHD QQGLKYPPT (SEQ ID NO: 48) (SEQ ID NO: 49)(SEQ ID NO: 50) hum 30C10 KSSQSLFWSESHMNYLA YASTRQS HHHYDSHT(SEQ ID NO: 26) (SEQ ID NO: 27) (SEQ ID NO: 28) hum 12E6 RTSQDIGNYLSGASNLED LQYDKYPNT (SEQ ID NO: 34) (SEQ ID NO: 35) (SEQ ID NO: 36)rat 1D10 KASQNINKYLD NADNLHT LQRESWPYT (SEQ ID NO: 56) (SEQ ID NO: 57)(SEQ ID NO: 58)

TABLE 4 Variable Heavy Chain CDR Sequences Antibody CDRH1 CDRH2 CDRH3hum GFSLPSHSVS IIWNQGGTDYNSAFKS NAYITDYYYENYFMDA 16C10 (SEQ ID(SEQ ID NO: 16) (SEQ ID NO: 19) NO: 14) hum GFSLPSHSVSIIWNX1GGTDYX2SAFKS NX3YITDYYYENYFX4DA 16C10 (SEQ ID X₁ = N, A, Q X₃ =M, L, A, K, F variable NO: 14) X₂ = N, A, Q X₄ = M, F, L (SEQ ID NO: 17)(SEQ ID NO: 20) hum 4C3 GFSLPSHSVS IIWNQGGTDYNSAFKS NAYITDYYYENYFMDA(SEQ ID (SEQ ID NO: 16) (SEQ ID NO: 19) NO: 14) hum GFSLTNNGVTEVSSGGSTDYNSALKS QEVFTGLLDY 23E12 (SEQ ID (SEQ ID NO: 52)(SEQ ID NO: 53) NO: 51) hum GFTFNNYWMT SVSNTGSSTYYPASVKG EGAYYLDY 30C10(SEQ ID (SEQ ID NO: 30) (SEQ ID NO: 31) NO: 29) hum 12E6 GFTFRDYYMVSISYEGSSIYYGESVKG HGFNPFDY (SEQ ID (SEQ ID NO: 38) (SEQ ID NO: 39)NO: 37) rat 1D10 GFSLTNYYVH GVWNDGDTSYNSVLRS EGREGFVGYYVMDA (SEQ ID(SEQ ID NO: 60) (SEQ ID NO: 61) NO: 59)

In general the CDRs for the humanized antibodies are identical to theCDRs of the parental rat antibodies, with the exception being CDRH2 andCDRH3 of hum 16C10 and hum 4C3, which each has a single amino acidchange from the respective rat CDRs. Although they were obtained asindependent clones, parental rat antibodies 16C10 and 4C3 are identicalin the sequence of the V_(H) region and differ in the V_(L) region onlyby a framework substitution (isoleucine at position 15 in 16C10 isvaline in 4C3). As a result, the CDRs are identical for these twoparental rat antibodies, and thus for their humanized forms.

Sequences are provided for humanized V_(L) regions of antibodies16C10/4C3 and 30C10 at SEQ ID NOs: 5 and 22, respectively. Sequences areprovided for humanized V_(H) regions of antibodies 16C10/4C3 and 30C10at SEQ ID NOs: 6 and 23, respectively. These humanized variable domainsmay be used to create full-length chimeric or humanized antibodies byadding the appropriate constant domain sequences. Other embodimentsinclude various other alterations in the CDR amino acid residues in the16C10 heavy chain, for example, as illustrated in Table 4. Withreference to the residue numbering of FIG. 1B, the alterations describedin Table 4 (and in SEQ ID NOs: 17 and 20) are N54A, N54Q, N60A, N60Q,M96L, M96A, M96K, M96F, M100hF, M100hL.

In one embodiment of the present invention, chimeric light and heavychains of antibody 16C10 are created by appending human constant domains(human kappa light chain and human IgG1 constant domain, respectively)to the C-terminus of the humanized V_(L) (SEQ ID NO: 5) and V_(H)regions (SEQ ID NO: 6). Sequences of chimeric 16C10 light and heavychains are provided at SEQ ID NOs: 9 and 10. In other embodiments,chimeric forms of antibodies 30C10 and 4C3 are created by fusing thesame constant domains from chimeric 16C10 to their respective humanizedV_(L) and V_(H) regions (SEQ ID NOs: 22 and 23 for 30C10; SEQ ID NOs: 5and 6 for 4C3). The chimeric form of humanized 4C3 would, of course, beidentical to the chimeric form of humanized 16C10.

In another embodiment, full length humanized antibodies are created bysubstituting framework residues (i.e. those amino acid residues in thevariable domain that are not part of a CDR) of the chimeric formsantibodies with human germline framework sequences, as described in moredetail in Example 3. The resulting antibodies retain only the CDRsequences from the rat antibodies, with the constant domains andframework sequences replaced by human-derived sequences. Full-lengthlight and heavy chains for humanized antibody 16C10, including signalsequences, are provided at SEQ ID NOs: 2 and 4, respectively. In otherembodiments, humanized forms of antibodies 4C3 and 23E12 are created byanalogy with the method described for 16C10, i.e. substituting theappropriate human framework sequences into the sequence of the chimericversions of these antibodies (described supra). See Example 3.

In a further embodiment, the full-length light and heavy chains of thehumanized antibodies of the present invention are cloned to have asignal peptide at their N-terminus to facilitate secretion from cellswhen the antibody is produced. In one embodiment, a 19 amino acid signalsequence is added to both the light and heavy chains of the humanized16C10 antibody (residues −19 to −1 of SEQ ID NOs: 2 and 4). DNAsequences of the full length light and heavy chains of humanized 16C10,with signal sequence added, are provided at SEQ ID NOs: 1 and 3. SuchDNA sequences can be cloned and expressed in any suitable expressionvector for production of the humanized antibodies of the presentinvention. In other embodiments, signal sequences may be added to thelight and heavy chains of humanized antibodies 30C10 and 4C3, asdescribed for antibody 16C10. In other embodiments, signal sequencepeptides are added that are different than the specific signal sequenceprovided in SEQ ID NOs: 1-4, depending on the intended method ofproduction of the antibodies. Such signal sequences may be obtained fromthe scientific literature, for example Choo et al. (2005) “SPdb—a signalpeptide database,” BMC Bioinformatics 6:249.

In yet other embodiments, different constant domains may be appended tothe humanized V_(L) and V_(H) regions provided herein. For example, if aparticular intended use of an antibody (or fragment) of the presentinvention were to call for altered effector functions, a heavy chainconstant domain other than IgG1 may be used. Although IgG1 antibodiesprovide for long half-life and for effector functions, such ascomplement activation and antibody-dependent cellular cytotoxicity, suchactivities may not be desirable for all uses of the antibody. In suchinstances an IgG4 constant domain, for example, may be used.

V. Affinity and Biological Activity of Humanized Anti-IL-17A

Antibodies having the characteristics identified herein as beingdesirable in a humanized anti-IL-17A antibody can be screened forinhibitory biologic activity in vitro, in vivo, or by measuring bindingaffinity. To screen for antibodies that bind to the same epitope onhuman IL-17A bound by an antibody of interest (e.g., those which blockbinding of the cytokine to its receptor), a routine cross-blocking assaycan be performed such as that described in ANTIBODIES, A LABORATORYMANUAL, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988).Alternatively, epitope mapping can be performed to determine whether theantibody binds an epitope of interest, e.g., as described in Champe etal. (1995) J. Biol. Chem. 270:1388-1394. Antibody affinities (e.g. forhuman IL-17A) may be determined using standard methods, including thosedescribed in Example 7. Preferred humanized antibodies are those whichbind human IL-17A with a K_(d) value of no more than about 100 nM(1×10⁻⁷M); preferably no more than about 10 nM; more preferably no morethan about 1 nM. Even more preferred are embodiments in which theantibodies have K_(d) values of no more than about 200 pM (2×10⁻¹⁰ M),100 pM, 50 pM, 20 pM, 10 pM, 5 pM or even 2 pM.

The antibodies, and fragments thereof, useful in the present compoundsand methods include, but are not limited to, biologically activeantibodies and fragments. As used herein, the term “biologically active”refers to an antibody or antibody fragment that is capable of bindingthe desired the antigenic epitope and directly or indirectly exerting abiologic effect. Typically, these effects result from the failure ofIL-17A to bind its receptor. As used herein, the term “specific” refersto the selective binding of the antibody to the target antigen epitope.Antibodies can be tested for specificity of binding by comparing bindingto IL-17A to binding to irrelevant antigen or antigen mixture under agiven set of conditions. An antibody is considered to be specific if itbinds to IL-17A with an affinity at least 10-fold, and preferably50-fold higher than its affinity for an irrelevant antigen or antigenmixture. An antibody that “specifically binds” to a protein comprisingIL-17A (or a fragment thereof) does not bind to proteins that do notcomprise the IL-17A-derived sequences, i.e. “specificity” as used hereinrelates to IL-17A specificity, and not any other sequences that may bepresent in the protein in question. For example, as used herein, anantibody that “specifically binds” to FLAG-hIL-17A, which is a fusionprotein comprising IL-17A and a FLAG® peptide tag, does not bind to theFLAG® peptide tag alone or when it is fused to a protein other thanIL-17A.

The data presented in the Examples below show (e.g. Example 7) thathumanized antibody 16C10 (including the N54Q and M96A substitutionsrelative to the parental rat heavy chain CDRs) has a high affinity forbinding to human IL-17A, with a K_(d) in the 1-10 pM range as determinedby KinExA analysis. In vitro activity assays, such as Ba/F3hIL-17Rc-GCSFR cell proliferation assay (Example 11), normal humandermal fibroblast (NHDF) assay (Example 9), and human rheumatoidarthritis (RA) synoviocyte assay (Example 8) confirm that hu16C10 is ahigh affinity antibody since the observed IC50 values were typicallyless than or equal to 50% of the concentration of hIL-17A present in theassay (100 pM, 1000 pM, and 1000 pM in the three assays, respectively).The bivalent character of the antibodies used in the experiments, andthe potential for IL-17A dimer formation, make it possible to achieve50% inhibition of a given concentration of IL-17A with less than 0.5molar equivalents of antibody. In vivo activity assays, such asadministration to mice exhibiting collagen-induced arthritis (Example16) and BAL neutrophil recruitment assay (Example 17) confirm theactivity of several of the anti-IL-17A antibodies of the presentinvention in animals. The in vitro and in vivo activity assays alsoconfirm that humanized antibody 16C10 is a neutralizing antibody, whichwas not known from the binding experiments alone.

The ability of several of the antibodies of the present invention tobind to cyno IL-17A as well as human IL-17A is advantageous because sucha potential therapeutic antibody can be used directly in cynomolgusmonkey for toxicology studies, rather than having to develop a separatecyno-specific antibody for such studies. The high affinity of several ofthe antibodies of the present invention is also advantageous in that mayreduce the required dosage in human (and other) subjects, which reducesthe likelihood of certain adverse reactions. In addition, the highaffinity may reduce the volume that must be administered to a subjectand reduce the cost of treatment.

The serum half-life of hu16C10 was measured in mouse and in cynomolgusmonkeys. In cyno, half-life after intravenous (iv) administration wasevaluated in a dose ranging study with 0.4, 4.0 and 40 mg/kg dosing.Serum concentrations of drug were measured periodically for 42 days. Thehalf-life for subcutaneous (sc) administration in cyno was determined at4 mg/kg dosing, which was also followed for 42 days. The half-life incynomolgus monkeys was 10-19 days iv and 28 days sc as measured by theterminal slope of the drug concentration versus time profile. Certainanomalous datapoints at higher dosings were excluded from the analysis.Similar experiments in mice showed that the hu16C10 antibody had ahalf-life of 13-25 days iv and 12-22 days sc.

Example 19 describes methods used to determine the epitope bound by anexemplary anti-IL-17A antibody of the present invention (16C10), i.e.residues in the region of L74-Y85 of human IL-17A (SEQ ID NO.: 40).Since the biological assay data presented herein demonstrate thatantibody 16C10 is a high affinity neutralizing antibody, otherantibodies that bind to the same epitope may also be expected to beneutralizing antibodies, and perhaps also have high binding affinity.The epitope as determined herein is obtained by functional measurements,rather than structure determinations, and the epitope reported hereinmay differ in detail from the epitope determined by structural methods.The epitope reported herein includes at least some, but not necessarilyall, of the amino acid residues that are important for antibody 16C10binding. The epitope bound by antibodies of the present invention mayalso be determined by other methods, such as cross-blocking experiments(see Example 12), or by structural methods such as X-ray crystalstructure determination. Additional antibodies binding to the sameepitope as antibody 16C10 may be obtained, for example, by screening ofantibodies raised against hIL-17A, or by immunization of an animal witha peptide comprising the epitope sequence.

V. Antibody Production

For recombinant production of the antibody, the nucleic acid encoding itis isolated and inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. DNA encoding themonoclonal antibody is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody). Many vectors are available. The vector components generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more marker genes, anenhancer element, a promoter, and a transcription termination sequence.In one embodiment, both the light and heavy chains of the humanizedanti-IL-17A antibody of the present invention are expressed from thesame vector, e.g. a plasmid or an adenoviral vector.

Antibodies of the present invention may be produced by any method knownin the art. In one embodiment, antibodies are expressed in mammalian orinsect cells in culture, such as chinese hamster ovary (CHO) cells,human embryonic kidney (HEK) 293 cells, mouse myeloma NSO cells, babyhamster kidney (BHK) cells, Spodoptera frugiperda ovarian (Sf9) cells.In one embodiment, antibodies secreted from CHO cells are recovered andpurified by standard chromatographic methods, such as protein A, cationexchange, anion exchange, hydrophobic interaction, and hydroxyapatitechromatography. Resulting antibodies are concentrated and stored in 20mM sodium acetate, pH 5.5.

In another embodiment, the antibodies of the present invention areproduced in yeast according to the methods described in WO2005/040395.Briefly, vectors encoding the individual light or heavy chains of anantibody of interest are introduced into different yeast haploid cells,e.g. different mating types of the yeast Pichia pastoris, which yeasthaploid cells are optionally complementary auxotrophs. The transformedhaploid yeast cells can then be mated or fused to give a diploid yeastcell capable of producing both the heavy and the light chains. Thediploid strain is then able to secret the fully assembled andbiologically active antibody. The relative expression levels of the twochains can be optimized, for example, by using vectors with differentcopy number, using transcriptional promoters of different strengths, orinducing expression from inducible promoters driving transcription ofthe genes encoding one or both chains.

In one embodiment, the respective heavy and light chains of a pluralityof different anti-IL-17A antibodies (the “original” antibodies) areintroduced into yeast haploid cells to create a library of haploid yeaststrains of one mating type expressing a plurality of light chains, and alibrary of haploid yeast strains of a different mating type expressing aplurality of heavy chains. These libraries of haploid strains can bemated (or fused as spheroplasts) to produce a series of diploid yeastcells expressing a combinatorial library of antibodies comprised of thevarious possible permutations of light and heavy chains. Thecombinatorial library of antibodies can then be screened to determinewhether any of the antibodies has properties that are superior (e.g.higher affinity for IL-17A) to those of the original antibodies. See.e.g., WO2005/040395.

In another embodiment, antibodies of the present invention are humandomain antibodies in which portions of an antibody variable domain arelinked in a polypeptide of molecular weight approximately 13 kDa. See,e.g., U.S. Pat. Publication No. 2004/0110941. Such single domain, lowmolecular weight agents provide numerous advantages in terms of ease ofsynthesis, stability, and route of administration.

VI. Pharmaceutical Compositions and Administration

To prepare pharmaceutical or sterile compositions of the anti-huIL-17Aantibodies of the present invention, the antibody is admixed with apharmaceutically acceptable carrier or excipient. See, e.g., Remington'sPharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, MackPublishing Company, Easton, Pa. (1984).

Formulations of therapeutic and diagnostic agents may be prepared bymixing with physiologically acceptable carriers, excipients, orstabilizers in the form of, e.g., lyophilized powders, slurries, aqueoussolutions or suspensions (see, e.g., Hardman, et al. (2001) Goodman andGilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, NewYork, N.Y.; Gennaro (2000) Remington: The Science and Practice ofPharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, etal. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications,Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical DosageForms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weinerand Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc.,New York, N.Y.). In one embodiment, anti-IL-17A antibodies of thepresent invention are diluted to an appropriate concentration in asodium acetate solution pH 5-6, and NaCl or sucrose is added fortonicity. Additional agents, such as polysorbate 20 or polysorbate 80,may be added to enhance stability.

Toxicity and therapeutic efficacy of the antibody compositions,administered alone or in combination with another agent, can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index (LD₅₀/ED₅₀). Antibodies exhibiting hightherapeutic indices are preferred. The data obtained from these cellculture assays and animal studies can be used in formulating a range ofdosage for use in human. The dosage of such compounds lies preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration.

The mode of administration is not particularly important. Suitableroutes of administration include oral, rectal, transmucosal, orintestinal administration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections. Administration can be carried out in a varietyof conventional ways, such as oral ingestion, inhalation, insufflation,topical application or cutaneous, transdermal, subcutaneous,intraperitoneal, parenteral, intra-arterial or intravenous injection.Intravenous administration to the patient is preferred.

Alternately, one may administer the antibody in a local rather thansystemic manner, for example, via injection of the antibody directlyinto an arthritic joint or pathogen-induced lesion characterized byimmunopathology, often in a depot or sustained release formulation.Furthermore, one may administer the antibody in a targeted drug deliverysystem, for example, in a liposome coated with a tissue-specificantibody, targeting, for example, arthritic joint or pathogen-inducedlesion characterized by immunopathology. The liposomes will be targetedto and taken up selectively by the afflicted tissue.

The administration regimen depends on several factors, including theserum or tissue turnover rate of the therapeutic antibody, the level ofsymptoms, the immunogenicity of the therapeutic antibody, and theaccessibility of the target cells in the biological matrix. Preferably,the administration regimen delivers sufficient therapeutic antibody toeffect improvement in the target disease state, while simultaneouslyminimizing undesired side effects. Accordingly, the amount of biologicdelivered depends in part on the particular therapeutic antibody and theseverity of the condition being treated. Guidance in selectingappropriate doses of therapeutic antibodies is available (see, e.g.,Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd,Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokinesand Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.) (1993)Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, MarcelDekker, New York, N.Y.; Baert, et al. (2003) New Engl. J. Med.348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973;Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al.(2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J.Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343:1594-1602).

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment. Generally, the dose begins with an amount somewhat less thanthe optimum dose and it is increased by small increments thereafteruntil the desired or optimum effect is achieved relative to any negativeside effects. Important diagnostic measures include those of symptomsof, e.g., the inflammation or level of inflammatory cytokines produced.Preferably, a biologic that will be used is derived from the samespecies as the animal targeted for treatment, thereby minimizing aninflammatory, autoimmune, or proliferative response to the reagent. Inthe case of human subjects, for example, chimeric, humanized and fullyhuman antibodies are preferred.

Antibodies, antibody fragments, and cytokines can be provided bycontinuous infusion, or by doses administered, e.g., daily, 1-7 timesper week, weekly, bi-weekly, monthly, bimonthly etc. Doses may beprovided intravenously, subcutaneously, topically, orally, nasally,rectally, intramuscular, intracerebrally, intraspinally, or byinhalation. A total weekly dose is generally at least 0.05 μg/kg bodyweight, more generally at least 0.2 μg/kg, 0.5 μg/kg, 1 μg/kg, 10 μg/kg,100 μg/kg, 0.25 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 5.0 mg/ml, 10 mg/kg, 25mg/kg, 50 mg/kg or more (see, e.g., Yang, et al. (2003) New Engl. J.Med. 349:427-434; Herold, et al. (2002) New Engl. J. Med. 346:1692-1698;Liu, et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456; Portielji,et al. (20003) Cancer Immunol. Immunother. 52:133-144). Doses may alsobe provided to achieve a pre-determined target concentration ofanti-IL-17A antibody in the subject's serum, such as 0.1, 0.3, 1, 3, 10,30, 100, 300 μg/ml or more. In other embodiments, a humanizedanti-IL-17A antibody of the present invention is administeredsubcutaneously or intravenously, on a weekly, biweekly or “every 4weeks” basis at 10, 20, 50, 80, 100, 200, 500, 1000 or 2500 mg/subject.

As used herein, “inhibit” or “treat” or “treatment” includes apostponement of development of the symptoms associated with a disorderand/or a reduction in the severity of the symptoms of such disorder. Theterms further include ameliorating existing uncontrolled or unwantedsymptoms, preventing additional symptoms, and ameliorating or preventingthe underlying causes of such symptoms. Thus, the terms denote that abeneficial result has been conferred on a vertebrate subject with adisorder, disease or symptom, or with the potential to develop such adisorder, disease or symptom.

As used herein, the terms “therapeutically effective amount”,“therapeutically effective dose” and “effective amount” refer to anamount of an IL-17A binding compound of the invention that, whenadministered alone or in combination with an additional therapeuticagent to a cell, tissue, or subject, is effective to prevent orameliorate one or more symptoms of a disease or condition or theprogression of such disease or condition. A therapeutically effectivedose further refers to that amount of the binding compound sufficient toresult in amelioration of symptoms, e.g., treatment, healing, preventionor amelioration of the relevant medical condition, or an increase inrate of treatment, healing, prevention or amelioration of suchconditions. When applied to an individual active ingredient administeredalone, a therapeutically effective dose refers to that ingredient alone.When applied to a combination, a therapeutically effective dose refersto combined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously. An effective amount of a therapeutic will result in animprovement of a diagnostic measure or parameter by at least 10%;usually by at least 20%; preferably at least about 30%; more preferablyat least 40%, and most preferably by at least 50%.

Methods for co-administration with a second therapeutic agent, e.g.,cytokine, another therapeutic antibody, steroid, chemotherapeutic agent,or antibiotic are well known in the art, see, e.g., Hardman, et al.(eds.) (2001) Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 10^(th) ed., McGraw-Hill, New York, N.Y.; Poole andPeterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: APractical Approach, Lippincott, Williams & Wilkins, Phila., PA; Chabnerand Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott,Williams & Wilkins, Phila., PA. The pharmaceutical composition of theinvention may also contain immunosuppressive or immunomodulating agents.Any suitable immunosuppressive agent can be employed, including but notlimited to anti-inflammatory agents, corticosteroids, cyclosporine,tacrolimus (i.e., FK-506), sirolimus, interferons, soluble cytokinereceptors (e.g., sTNRF and sIL-1R), agents that neutralize cytokineactivity (e.g., inflixmab, etanercept), mycophenolate mofetil,15-deoxyspergualin, thalidomide, glatiramer, azathioprine, leflunomide,cyclophosphamide, methotrexate, and the like. The pharmaceuticalcomposition can also be employed with other therapeutic modalities suchas phototherapy and radiation.

The IL-17A binding compounds of the present invention can also be usedin combination with one or more antagonists of other cytokines (e.g.antibodies), including but not limited to, IL-23, IL-113, IL-6 andTGF-β. See, e.g., Veldhoen (2006) Immunity 24:179-189; Dong (2006) Nat.Rev. Immunol. 6(4):329-333. In various embodiments, an IL-17A bindingcompound of the invention is administered before, concurrently with, orafter administration of the another antagonist or antagonists. In oneembodiment, an IL-17A binding compound of the present invention is usedin treatment of the acute early phase of an adverse immune response(e.g. MS, Crohn's Disease) alone or in combination with an IL-23antagonist. In the latter case, the IL-17A binding compound may begradually decreased and treatment with the antagonist of IL-23 alone iscontinued to maintain suppression of the adverse response.Alternatively, antagonists to IL-1β, IL-6 and/or TGF-β may beadministered concurrently, before or after an IL-17A binding compound ofthe present invention. See Cua and Kastelein (2006) Nat. Immunol.7:557-559; Tato and O'Shea (2006) Nature 441:166-168; Iwakura andIshigame (2006) J. Clin. Invest. 116:1218-1222.

Typical veterinary, experimental, or research subjects include monkeys,dogs, cats, rats, mice, rabbits, guinea pigs, horses, and humans.

VII. Uses

The present invention provides methods for using engineered anti-IL-17Aantibodies for the treatment and diagnosis of inflammatory disorders andconditions, as well as autoimmune and proliferative disorders. Methodsare provided for the diagnosis, prevention or treatment of inflammatorybowel disease (IBD), multiple sclerosis (MS), chronic obstructivepulmonary disease (COPD), cystic fibrosis (CF), psoriasis, systemicscleroderma, allograft rejection, autoimmune myocarditis and peritonealadhesions (see, e.g., Chung et al. (2002) J. Exp. Med. 195:1471-78).

Psoriasis

The skin serves as an important boundary between the internal milieu andthe environment, preventing contact with potentially harmful antigens.In the case of antigen/pathogen penetration, an inflammatory response isinduced to eliminate the antigen. This response leads to a dermalinfiltrate that consists predominantly of T cells, polymorphonuclearcells, and macrophages (see, e.g., Williams and Kupper (1996) Life Sci.,58:1485-1507.) Normally, this inflammatory response, triggered by thepathogen, is under tight control and will be halted upon elimination ofthe pathogen.

In certain cases this inflammatory response occurs without externalstimuli and without proper controls, leading to cutaneous inflammation.The present invention provides methods for treating and diagnosingcutaneous inflammation. Cutaneous inflammation, the result of thecellular infiltrate noted above as well as the secreted cytokines fromthese cells, encompasses several inflammatory disorders such ascicatricial pemphigoid, scleroderma, hidradenitis suppurativa, toxicepidermal necrolysis, acne, osteitis, graft vs. host disease (GvHD),pyroderma gangrenosum, and Behcet's Syndrome (see, e.g., Willams andGriffiths (2002) Clin. Exp. Dermatol., 27:585-590). The most common formof cutaneous inflammation is psoriasis.

Psoriasis is characterized by T cell mediated hyperproliferation ofkeratinocytes coupled with an inflammatory infiltrate. The disease hascertain distinct overlapping clinical phenotypes including chronicplaque lesions, skin eruptions, and pustular lesions (see, e.g.,Gudjonsson et al. (2004) Clin Exp. Immunol. 135:1-8). Approximately 10%of psoriasis patients develop arthritis. The disease has a strong butcomplex genetic predisposition, with 60% concordance in monozygotictwins.

The typical psoriatic lesion is a well defined erythematous plaquecovered by thick, silvery scales. The inflammation andhyperproliferation of psoriatic tissue is associated with a differenthistological, antigenic, and cytokine profile than normal skin. Amongthe cytokines associated with psoriasis are: TNFα, IL-19, IL-18, IL-15,IL-12, IL-7, IFNγ, IL-17A and IL-23 (see Gudjonsson et al., supra).IL-17A has been detected in psoriatic skin.

Anti-IL-17A antibodies of the present invention, either alone or incombination with other agents, may be used in prevention, treatment,diagnosis and prediction of psoriasis flare-ups. Use of anti-IL-17Aantibodies in prediction and treatment of psoriatic outbreaks isdescribed in commonly assigned U.S. Patent Application Publication2005/0287593 and PCT Patent Publication WO 2005/108616, the disclosuresof which are hereby incorporated by reference in their entireties.

Rheumatoid Arthritis (RA)

RA is a progressive, systemic disease characterized by inflammation ofthe synovial joints affecting about 0.5% of the world's population.Emery (2006) BMJ 332:152-155. Joint inflammation can lead to deformity,pain, stiffness and swelling, and ultimately to irreversibledeterioration of the joint. Affected joints include knees, elbows, neckand joints of the hands and feet. Conventional treatment involves use ofNSAIDs to alleviate symptoms, followed by administration of diseasemodifying antirheumatic drugs (DMARDs) such as gold, penicillamine,sulfasalazine and methotrexate. Recent advances include treatment withTNF-α inhibitors, including monoclonal antibodies, such as infliximab,adalumimab and golimumab, and receptor fusion proteins, such asetanercept. Treatment with these TNF-α inhibitors dramatically reducesstructural damage from the disease.

The anti-IL-17A antibodies of the present invention may be used to treatRA in subjects in need of such treatment. Example 16 describesexperiments involving the collagen-induced arthritis (CIA) model of RA,for which data are presented at FIGS. 3A-3D, and Table 15. The resultsshow a reduction in the fraction of paws with high disease severityscores in animals treated with an anti-IL-17A antibody of the presentinvention as compared with diluent and isotype controls.

The anti-IL-17A antibodies of the present invention may also be combinedwith other treatments for RA, e.g. methotrexate, azathioprine,cyclophosphamide, steroids, mycophenolate mofetil, NSAIDs, or TNF-αinhibitors (antibodies or receptor fragments).

In one embodiment, the anti-IL-17A antibodies of the present inventionare used to treat human subjects who have not previously respondedadequately to treatment with DMARDs alone. In another embodiment,treatment with the anti-IL-17A antibodies of the present invention isbegun early in the course of disease, without requiring prior failure ofDMARD therapy. Such early intervention may be appropriate, for example,once the safety of the antibody therapy has been firmly established.

Clinical improvement is measured by determining the ACR score, asdescribed in more detail in Example 18. In various embodiments, ACRscores of 20, 50, and 70 are the desired endpoint, and these endpointsmay be assessed at any appropriate point in the course of treatment,such as 5, 10, 15, 24, 40, 50 or more weeks.

Multiple Sclerosis (MS)

MS is thought to be an autoimmune disease of the central nervous system(CNS) involving loss of myelin from nerve fibers, resulting in plaquesor lesions. The most common form is relapsing/remitting MS in which welldefined symptomatic flare-ups occur, followed by periods of partial orcomplete remission. Conventional treatment options includeinterferon-β-1a and -1b, mitoxantrone, the tetrapeptide glatirameracetate, therapeutic alpha-4-integrin-specific antibodies (natalizumab),or small molecule antagonists of alpha-4-integrin (e.g. those disclosedat WO2003/084984).

The anti-IL-17A antibodies of the present invention may be used to treatMS in subjects in need of such treatment. The anti-IL-17A antibodies mayalso be combined with other treatments for MS, e.g. interferon-β,interferon-α, steroids or alpha-4-integrin-specific antibodies.

Inflammatory Bowel Disease (IBD)

IBD is the name for a group of disorders (e.g. Crohn's disease andulcerative colitis) in which the intestines become inflamed, resultingin abdominal cramps and pain, diarrhea, weight loss and intestinalbleeding. IBD affects over 600,000 Americans. Conventional treatmentoptions include sulfasalazine, corticosteroids (e.g. prednisone), immunesystem suppressors such as azathioprine and mercaptopurine, or anantibiotic (e.g. metronidazole) for Crohn's disease. Therapeuticmonoclonal antibody treatments include etanercept, natalizumab andinfliximab.

The anti-IL-17A antibodies of the present invention may be used to treatIBD in subjects in need of such treatment. Yen et al. (2006) J. Clin.Invest. 116:1310-1316; Fujimo et al. (2003) Gut 52:65-70. Theanti-IL-17A antibodies of the present invention may also be combinedwith other treatments for IBD, e.g. IL-10 (see U.S. Pat. Nos. 5,368,854,7,052,686), steroids and sulfasalazine.

In other embodiments, antibodies of the present invention that do notblock binding of IL-17A to its receptor (e.g. non-neutralizing antibody12E6) are used therapeutically to stabilize IL-17A in subjects in needto prolonged IL-17A activity. Such subjects include patients sufferingfrom infections or cancers.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The invention is defined by the terms of theappended claims, along with the full scope of equivalents to which suchclaims are entitled. The specific embodiments described herein,including the following examples, are offered by way of example only,and do not by their details limit the scope of the invention.

Example 1 General Methods

Standard methods in molecular biology are described (Maniatis, et al.(1982) Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Sambrook and Russell (2001)Molecular Cloning, 3^(rd) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Wu (1993) Recombinant DNA, Vol. 217, AcademicPress, San Diego, Calif.). Standard methods also appear in Ausbel, etal. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wileyand Sons, Inc. New York, N.Y., which describes cloning in bacterialcells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast(Vol. 2), glycoconjugates and protein expression (Vol. 3), andbioinformatics (Vol. 4).

Methods for protein purification including immunoprecipitation,chromatography, electrophoresis, centrifugation, and crystallization aredescribed (Coligan, et al. (2000) Current Protocols in Protein Science,Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis,chemical modification, post-translational modification, production offusion proteins, glycosylation of proteins are described (see, e.g.,Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2,John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) CurrentProtocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY,N.Y., pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for LifeScience Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech(2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production,purification, and fragmentation of polyclonal and monoclonal antibodiesare described (Coligan, et al. (2001) Current Protocols in Immunology,Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999)Using Antibodies, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Harlow and Lane, supra). Standard techniques forcharacterizing ligand/receptor interactions are available (see, e.g.,Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, JohnWiley, Inc., New York).

Monoclonal, polyclonal, and humanized antibodies can be prepared (see,e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ.Press, New York, N.Y.; Kontermann and Dubel (eds.) (2001) AntibodyEngineering, Springer-Verlag, New York; Harlow and Lane (1988)Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., pp. 139-243; Carpenter, et al. (2000) J.Immunol. 165:6205; He, et al. (1998) J. Immunol. 160:1029; Tang et al.(1999) J. Biol. Chem. 274:27371-27378; Baca et al. (1997) J. Biol. Chem.272:10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote andWinter (1992) J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).

An alternative to humanization is to use human antibody librariesdisplayed on phage or human antibody libraries in transgenic mice(Vaughan et al. (1996) Nature Biotechnol. 14:309-314; Barbas (1995)Nature Medicine 1:837-839; Mendez et al. (1997) Nature Genetics15:146-156; Hoogenboom and Chames (2000) Immunol. Today 21:371-377;Barbas et al. (2001) Phage Display: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; Kay et al. (1996)Phage Display of Peptides and Proteins: A Laboratory Manual, AcademicPress, San Diego, Calif.; de Bruin et al. (1999) Nature Biotechnol.17:397-399).

Single chain antibodies and diabodies are described (see, e.g., Maleckiet al. (2002) Proc. Natl. Acad. Sci. USA 99:213-218; Conrath et al.(2001) J. Biol. Chem. 276:7346-7350; Desmyter et al. (2001) J. Biol.Chem. 276:26285-26290; Hudson and Kortt (1999) J. Immunol. Methods231:177-189; and U.S. Pat. No. 4,946,778). Bifunctional antibodies areprovided (see, e.g., Mack, et al. (1995) Proc. Natl. Acad. Sci. USA92:7021-7025; Carter (2001) J. Immunol. Methods 248:7-15; Volkel, et al.(2001) Protein Engineering 14:815-823; Segal, et al. (2001) J. Immunol.Methods 248:1-6; Brennan, et al. (1985) Science 229:81-83; Raso, et al.(1997) J. Biol. Chem. 272:27623; Morrison (1985) Science 229:1202-1207;Traunecker, et al. (1991) EMBO J. 10:3655-3659; and U.S. Pat. Nos.5,932,448, 5,532,210, and 6,129,914).

Bispecific antibodies are also provided (see, e.g., Azzoni et al. (1998)J. Immunol. 161:3493; Kita et al. (1999) J. Immunol. 162:6901; Merchantet al. (2000) J. Biol. Chem. 74:9115; Pandey et al. (2000) J. Biol.Chem. 275:38633; Zheng et al. (2001) J. Biol. Chem. 276:12999; Propst etal. (2000) J. Immunol. 165:2214; Long (1999) Ann. Rev. Immunol. 17:875).

Purification of antigen is not necessary for the generation ofantibodies. Animals can be immunized with cells bearing the antigen ofinterest. Splenocytes can then be isolated from the immunized animals,and the splenocytes can fused with a myeloma cell line to produce ahybridoma (see, e.g., Meyaard et al. (1997) Immunity 7:283-290; Wrightet al. (2000) Immunity 13:233-242; Preston et al., supra; Kaithamana etal. (1999) J. Immunol. 163:5157-5164).

Antibodies will usually bind with at least a K_(d) of about 10⁻⁶ M,typically at least 10⁻⁷ M, more typically at least 10⁻⁸ M, preferably atleast about 10⁻⁹ M, and more preferably at least 10⁻¹⁰ M, and mostpreferably at least 10⁻¹¹ M (see, e.g., Presta et al. (2001) Thromb.Haemost. 85:379-389; Yang et al. (2001) Crit. Rev. Oncol. Hematol.38:17-23; Carnahan et al. (2003) Clin. Cancer Res. (Suppl.)9:3982s-3990s).

Antibodies can be conjugated, e.g., to small drug molecules, enzymes,liposomes, polyethylene glycol (PEG). Antibodies are useful fortherapeutic, diagnostic, kit or other purposes, and include antibodiescoupled, e.g., to dyes, radioisotopes, enzymes, or metals, e.g.,colloidal gold (see, e.g., Le Doussal et al. (1991) J. Immunol.146:169-175; Gibellini et al. (1998) J. Immunol. 160:3891-3898; Hsingand Bishop (1999) J. Immunol. 162:2804-2811; Everts et al. (2002) J.Immunol. 168:883-889).

Methods for flow cytometry, including fluorescence activated cellsorting (FACS), are available (see, e.g., Owens, et al. (1994) FlowCytometry Principles for Clinical Laboratory Practice, John Wiley andSons, Hoboken, N.J.; Givan (2001) Flow Cytometry, 2^(nd) ed.;Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical Flow Cytometry, JohnWiley and Sons, Hoboken, N.J.). Fluorescent reagents suitable formodifying nucleic acids, including nucleic acid primers and probes,polypeptides, and antibodies, for use, e.g., as diagnostic reagents, areavailable (Molecular Probes (2003) Catalogue, Molecular Probes, Inc.,Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).

Standard methods of histology of the immune system are described (see,e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology andPathology, Springer Verlag, New York, N.Y.; Hiatt, et al. (2000) ColorAtlas of Histology, Lippincott, Williams, and Wilkins, Phila, Pa.;Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, NewYork, N.Y.).

Software packages and databases for determining, e.g., antigenicfragments, leader sequences, protein folding, functional domains,glycosylation sites, and sequence alignments, are available (see, e.g.,GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, Md.); GCG WisconsinPackage (Accelrys, Inc., San Diego, Calif.); DeCypher® (TimeLogic Corp.,Crystal Bay, Nev.); Menne, et al. (2000) Bioinformatics 16: 741-742;Menne, et al. (2000) Bioinformatics Applications Note 16:741-742; Wren,et al. (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne(1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res.14:4683-4690).

Example 2 Rat Anti Human IL-17A Monoclonal Antibodies

Monoclonal antibodies to human IL-17A were obtained as follows. Eightweek old female Lewis rats (Harlan Sprague Dawley, Indianapolis, Ind.,USA) were given a series of injections of recombinant human IL-17A(rhIL-17A) that had been expressed from adenoviral vectors in HEK 293cells. The injections were given at days 0, 14, 32, 46, and 83.

The day 0 injection was a subcutaneous (sc) injection of 50 μg rhIL-17A,accompanied by intraperitoneal (ip) injection of Freund's CompleteAdjuvant. Day 14, 32 and 46 sc injections of 25 μg rhIL-17A wereaccompanied by ip injection of Freund's Incomplete Adjuvant. The day 83injection was a combination of an ip injection of 20 μg rhIL-17A inFreund's Incomplete Adjuvant and an intravenous (iv) tail vein injectionof rhIL-17A in saline.

A test bleed was performed at day 53. Fusion of rat splenocytes wasperformed on day 87, using 1.6×10⁸ splenocytes and 1.8×10⁸ myeloma cellsdivided into in thirty 96-well plates, giving a total of 1.13×10⁵ totalcells per well.

Primary screening of the resulting monoclonal antibodies (thousands) wasperformed by indirect rhIL-17A ELISA (see Example 5). Secondary screenson the resulting antibodies included neutralization of rhIL-17A-inducedexpression of murine IL-6 by ST2 (mouse stromal) cells andneutralization of rhIL-17A-induced proliferation of Ba/F3hIL-17Rc:mGCSFR cells (see Example 11). Approximately eleven of themonoclonals were studied further after the first and second screens.Subsequent experiments were performed to confirm that the candidateantibodies were able to bind to native huIL-17A to ensure that theywould be useful in various therapeutic, diagnostic and/or researchpurposes. Such screening may be done using binding assays (such asindirect ELISA or sandwich ELISA), by in vitro activity assay, or by invivo activity assay, examples of which are provided herein.

Example 3 Humanization of Rat Anti Human IL-17A Antibodies

The humanization of rat anti human IL-17A monoclonal antibody 16C10 wasperformed essentially as described in WO 2005/047324 and WO 2005/047326,the disclosures of which are hereby incorporated by reference in theirentireties. Briefly, human constant domains were used to replace theparental (rat) constant domains, and human germline sequences homologousto the rat variable domain sequences were selected and used to provide ahuman framework for the rat CDRs, as described in more detail below.

Procedure for Selection of Human Germline Framework Sequences

The following steps are used in selecting the appropriate germlineframework sequences in humanizing the anti-human IL-17A antibodies ofthe present invention.

1) Clone and sequence non-human V_(L) and V_(H) domains and determineamino acid sequence.

Heavy Chain

2) Compare the non-human V_(H) sequence to a group of five human V_(H)germline amino acid sequences; one representative from subgroups IGHV1and IGHV4 and three representatives from subgroup IGHV3. The V_(H)subgroups are listed in M.-P. Lefranc (2001) “Nomenclature of the HumanImmunoglobulin Heavy (IGH) Genes”, Experimental and ClinicalImmunogenetics, 18:100-116. Comparison to the five germline sequences isperformed as follows:

-   -   A) Assign the non-human V_(H) sequence residue numbers according        to Kabat et al. (1991).    -   B) Align the non-human V_(H) sequence with each of the five        human germline sequences. Since the V genes only comprise V_(H)        residues 1-94, only these residues are considered in the        alignment.    -   C) Delineate the complementarity-determining (CDR) and framework        (FR) regions in the sequence. CDR and FR are defined as a        combination of the definitions provided in Kabat et al. (1991)        (Id.) and Chothia and Lesk (1987) “Canonical Structures for the        Hypervariable Regions of Immunoglobulins”, Journal of Molecular        Biology, 196:901-917. The definition is thus: V_(H) CDR1=26-35,        CDR2=50-65, CDR3=95-102.    -   D) For each listed residue position below (Table 1), assign        numerical score at each residue position for which the non-human        and human sequences are IDENTICAL:

TABLE 1 Residue # Score Reason 2 4 Affects CDR-H1,3* 4 3 AffectsCDR-H1,3 24 3 Affects CDR-H1 26 4 Affects CDR-H1* 27 4 Affects CDR-H1,3*29 4 Affects CDR-H1* 34 4 Affects CDR-H1* 35 2 VH/VL interface 37 2VH/VL interface 39 2 VH/VL interface 44 2 VH/VL interface 45 2 VH/VLinterface 47 4 VH/VL interface, CDRL3 48 3 Affects CDR-H2 49 3 AffectsCDR-H2 50 2 VH/VL interface 51 3 Affects CDR-H2 58 2 VH/VL interface 593 Affects CDR-H2 60 2 VH/VL interface 63 3 Affects CDR-H2 67 3 AffectsCDR-H2 69 3 Affects CDR-H2 71 4 Affects CDR-H2* 73 3 Affects CDR-H1 76 3Affects CDR-H1 78 3 Affects CDR-H1 91 2 VH/VL interface 93 3 AffectsCDR-H3 94 4 Affects CDR-H3* max 89 *Noted as affecting CDR conformationin C. Chothia et al. (1989) “Conformations of ImmunoglobulinHypervariable Regions”, Nature 342: 877-883.

-   -   E) Add all residue position scores. Acceptor germline sequence        is the one with the highest total score. In a case where two or        more germline sequences have identical scores, then:        -   1) Among the following residue positions add 1 to the total            for each position where the non-human and human sequences            are IDENTICAL: 1, 3, 5-23, 25, 36, 38, 40-43, 46, 66, 68,            70, 72, 74, 75, 77, 79-90, 92 (max 49).        -   2) Acceptor germline sequence is the one with the highest            total score. If two or more germline sequences still have            identical scores, either one is acceptable as acceptor.            Light Chain

III) If the V_(L) sequence is a member of the kappa subclass of V_(L),compare non-human V_(L) sequence to a group of four human V_(L) kappagermline amino acid sequences. The group of four is comprised of onerepresentative from each of four established human V_(L) subgroupslisted in Barbie and Lefranc (1998) “The Human Immunoglobulin KappaVariable (IGKV) Genes and Joining (IGKJ) Segments”, Experimental andClinical Immunogenetics, 15:171-183, and M.-P. Lefranc (2001)“Nomenclature of the Human Immunoglobulin Kappa (IGK) Genes”,Experimental and Clinical Immunogenetics, 18:161-174. The four subgroupsalso correspond to the four subgroups listed in Kabat et al. (1991) atpp. 103-130. Comparison to the four germline sequences is performed asfollows:

-   -   A) Assign the non-human V_(L) sequence residue numbers according        to Kabat et al. (1991).    -   B) Align the non-human V_(L) sequence with each of the four        human germline sequences. Since the V genes only comprise V_(L)        residues 1-95, only these residues are considered in the        alignment.    -   C) Delineate the complementarity-determining (CDR) and framework        (FR) regions in the sequence. CDR and FR are defined as a        combination of the definitions provided in Kabat et al. (1991)        and Chothia and Lesk (1987) “Canonical Structures for the        Hypervariable Regions of Immunoglobulins”, Journal of Molecular        Biology, 196:901-917. The definition is thus: V_(L) CDR1=24-34,        CDR2=50-56, CDR3=89-97.    -   D) For each listed residue position below (Table 2), assign        numerical score at each residue position for which the non-human        and human sequences are IDENTICAL:

TABLE 2 Residue # Score Reason 2 4 Affects CDR-L1,3* 4 3 AffectsCDR-L1,3 25 4 Affects CDR-L1* 29 4 Affects CDR-L1,3* 33 4 AffectsCDR-L1,3* 34 2 VL/VH interface 36 2 VL/VH interface 38 2 VL/VH interface43 2 VL/VH interface 44 2 VL/VH interface 46 4 VL/VH interface, CDR-H347 3 Affects CDR-L2 48 4 Affects CDR-L2* 49 2 VL/VH interface 55 2 VL/VHinterface 58 3 Affects CDR-L2 62 3 Affects CDR-L2 64 4 Affects CDR-L2*71 4 Affects CDR-L1* 87 2 VL/VH interface 89 2 VL/VH interface 90 4Affects CDR-L3* 91 2 VL/VH interface 94 2 VL/VH interface 95 4 AffectsCDR-L3* *Noted as affecting CDR conformation in C. Chothia et al.“Conformations of Immunoglobulin Hypervariable Regions”, Nature 342:877-883, 1989.

-   -   E) Add all residue position scores. Acceptor germline sequence        is the one with the highest total score. In a case where two or        more germline sequences have identical scores, then:        -   1) Among the following residue positions add 1 to the total            for each position where the non-human and human sequences            are IDENTICAL: 1, 3, 5-23, 35, 37, 39-42, 57, 59-61, 63,            65-70, 72-86, 88.        -   2) Acceptor germline sequence is the one with the highest            total score. If two or more germline sequences still have            identical scores, either one is acceptable as acceptor.

If the V_(L) sequence is a member of the lambda subclass of V_(L), ananalogous procedure is performed using human V_(L) lambda germline aminoacid sequences from the literature sources cited above.

Humanization of Anti-Human IL-17A Antibodies

With regard to modification of the constant domains, the variable lightand heavy domains of antibody 16C10 (rat anti-human IL-17A IgG1) werecloned and fused to a human kappa light chain (CL domain) and human IgG1heavy chain (CH1-hinge-CH2-CH3), respectively. This combination of therat variable domains and human constant domains comprises a chimericversion of antibody 16C10. The sequences of the light and heavy chainsof this chimeric 16C10 are provided at SEQ ID NOs: 9 and 10,respectively.

With regard to modification of the framework regions of the variabledomains, the amino acid sequence of the V_(H) domain of antibody 16C10was compared to a group of five human V_(H) germline amino acidsequences; one representative from subgroups IGHV1 and IGHV4 and threerepresentatives from subgroup IGHV3. The V_(H) subgroups are listed inM.-P. Lefranc, “Nomenclature of the Human Immunoglobulin Heavy (IGH)Genes,” Experimental and Clinical Immunogenetics, 18:100-116, 2001.Antibody 16C10 scored highest against human heavy chain germline DP-71in subgroup IV.

The V_(L) sequence of 16C10 was of the kappa subclass. This sequence wascompared to a group of four human V_(L) kappa germline amino acidsequences. The group of four is comprised of one representative fromeach of four established human V_(L) subgroups listed in V. Barbie &M.-P. Lefranc, “The Human Immunoglobulin Kappa Variable (IGKV) Genes andJoining (IGKJ) Segments”, Experimental and Clinical Immunogenetics,15:171-183, 1998 and M.-P. Lefranc, “Nomenclature of the HumanImmunoglobulin Kappa (IGK) Genes”, Experimental and ClinicalImmunogenetics, 18:161-174, 2001. The four subgroups also correspond tothe four subgroups listed in Kabat et al. (1991) at pp. 103-130.Antibody 16C10 scored highest against human light chain germline Z-A19in subgroup II.

Once the desired germline framework sequences were determined, a plasmidencoding the full-length humanized variable heavy and light chains wasgenerated. Substitution of human framework residues in place of theframework residues of the parental rat antibody 16C10 can be viewedequivalently as the grafting of the rat 16C10 CDRs onto the humanframework sequences. The resulting antibody is referred to herein as“16C10wt”, with the “wt” designating the presence of the same CDRs asthe parental rat 16C10, as distinguished from the optimized CDRs (havingtwo single amino acid alterations) discussed below. Both the light andheavy chain variable domains were codon optimized, synthesized andinserted onto constant domains to provide for potentially optimalexpression. Codon optimization, which may improve expression of clonedantibodies, is purely optional.

In addition to the substitution of human constant domain and frameworksequences, the humanized 16C10 wt antibody was also modified at two CDRresidues to provide for greater chemical stability of the finalhumanized antibody. The two changes are represented by bolded amino acidresidues in the “hu16C10” V_(H) sequence shown in FIG. 1B. Withreference to the Kabat numbering used in FIG. 1B, residue 54 of CDR2 waschanged from N (asparagine) in the rat antibody to Q (glutamine) in thehumanized antibody to reduce the potential for formation of isoaspartateat the NG sequence at residues 54-55. Isoaspartate formation maydebilitate or completely abrogate binding of an antibody to its targetantigen. Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734. Inaddition, residue 96 of CDR3 was changed from M (methionine) in the ratantibody to A (alanine) in the humanized antibody to reduce thepossibility that the methionine sulfur would oxidize, which could reduceantigen binding affinity and also contribute to molecular heterogeneityin the final antibody preparation. Id. These single-residuemodifications can be represented as N54Q and M96A. The final humanized16C10 antibody disclosed herein comprises these two substitutionsrelative to the parental rat 16C10 CDRs.

In another embodiment of the present invention, the chimeric (nothumanized) 16C10 antibody is altered to incorporate the twosingle-residue modifications described above for the humanized form,i.e. N54Q and M96A.

The amino acid sequences of the light and heavy chains of humanizedantibody 16C10 (hu 16C10) are provided at FIGS. 2A and 2B respectively,and at SEQ ID NOs: 2 and 4 (which include signal sequences). Oneembodiment of nucleotide sequences encoding the light and heavy chainsof hu 16C10 are shown in SEQ ID NOs:1 and 3. Another embodiment ofnucleotide sequences encoding the light and heavy chains of hu 16C10 areshown in FIG. 5A (SEQ ID NO:62) and FIG. 5B (SEQ ID NO:63).

In the interest of clarity with regard to nomenclature, it is importantto recognize that the Kabat numbering system includes non-numericalamino acid residue designations (e.g. V_(H) residues 83a, 83b, 83c) toaccommodate variations in the lengths of CDRs and framework regionsamong various antibodies. Although this numbering system is advantageousin allowing easy reference to corresponding amino acid residues amongvarious antibodies with CDRs of different lengths, it can result inconflicting designations for specific amino acid residues when comparedwith strict sequential-numeric sequence numbering (e.g. sequencelistings). Amino acid residue designations herein are made withreference to the relevant sequence listing unless otherwise noted, forexample by reference to “Kabat numbering”.

As an additional point of clarification with regard to nomenclature, SEQID NOs: 2 and 4 (humanized 16C10) include the sequences of N-terminalsignal peptides (the first 19 residues of each), which amino acids areremoved in the mature form of the antibody. SEQ ID NOs: 1, 3, 62 and 63include 57 nucleotides encoding the signal sequences. As used herein, a“mature” form of a protein refers to the protein without the signalsequence.

Humanized antibody 4C3 is created by methods analogous to thosedescribed above for antibody 16C10. Because the parental rat 4C3antibody differed only at a single amino acid residue in the frameworkregion of the light chain, and such framework regions are replaced withhuman germline framework sequences during humanization, the ultimatehumanized 4C3 antibody sequence is identical to the sequence ofhumanized 16C10 antibody.

Humanized antibody 30C10 is also created by methods analogous to thosedescribed above for antibody 16C10. In determining the proper humanframework sequences to be used, the parental rat 30C10 antibody scoreshighest against human heavy chain germline DP-46 in subgroup III andhuman light chain germline Z-A19 in subgroup II, so those frameworksequences are substituted for the rat framework sequences. The humanized30C10 V_(L) and V_(H) sequences are provided at SEQ ID NOs: 22 and 23,respectively. In other embodiments, one or more methionine residues inthe CDRs of rat 30C10 are mutated to avoid the potential of oxidation ofthe methionine sulfur in the humanized 30C10 antibody. Specifically,heavy chain residue 34 (in CDRH1) and/or light chain residue 30f (Kabatnumbering, see FIG. 1A) are changed from methionine to another aminoacid, e.g. alanine. Such antibodies are subsequently screened to ensurethat the methionine substitution does not decrease IL-17A bindingaffinity to unacceptable levels.

Chimeric, humanized, and signal sequence-containing versions of antibody12E6 are created using the methods described herein, by analogy withpreparation of such antibodies based on parental rat antibody 16C10.Light and heavy chain CDRs for parental rat antibody 12E6 are providedat SEQ ID NOs: 34-36 and 37-39. Human constant domain and variabledomain framework sequences are introduced as described above. In oneembodiment, heavy chain residue 34 (in CDRH1) is changed from amethionine to another amino acid, e.g. alanine, to avoid the potentialof oxidation of the methionine sulfur in the humanized 12E6 antibody.The resulting antibodies are subsequently screened to ensure that themethionine substitution does not decrease IL-17A binding affinity tounacceptable levels.

Chimeric, humanized, and signal sequence-containing versions of antibody23E12 are created using the methods described herein, by analogy withpreparation of such antibodies based on parental rat antibody 16C10.Light and heavy chain variable domain sequences for the parental ratantibody 23E12 are provided at SEQ ID NOs: 44 and 46 (DNA), and 45 and47 (amino acid). CDRs for parental rat antibody 23E12 are provided atSEQ ID NOs: 48-50 (light chain) and 51-53 (heavy chain). Human constantdomain and variable domain framework sequences are introduced into theparental rat antibodies as described above.

Example 4 Fully Human Anti-IL-17A Antibodies

Fully human anti-IL-17A monoclonal antibodies are generated usingtransgenic mice carrying parts of the human immune system rather thanthe mouse system. These transgenic mice, referred to herein as “HuMAb”mice, contain human immunoglobulin gene miniloci that encodeunrearranged human heavy (μ and γ) and κ light chain immunoglobulinsequences, together with targeted mutations that inactivate theendogenous μ and κ chain loci (Lonberg et al. (1994) Nature368(6474):856-859). Accordingly, the mice exhibit reduced expression ofmouse IgM or κ, and in response to immunization, the introduced humanheavy and light chain transgenes undergo class switching and somaticmutation to generate high affinity human IgG κ monoclonal antibodies(Lonberg et al. (1994), supra; reviewed in Lonberg (1994) Handbook ofExperimental Pharmacology 113:49-101; Lonberg et al. (1995) Intern. Rev.Immunol. 13:65-93, and Harding et al. (1995) Ann. N Y. Acad. Sci.764:536-546). The preparation of HuMab mice is commonly known in the artand is described, for example, in Taylor et al. (1992) Nucleic AcidsResearch 20:6287-6295; Chen et al. (1993) International Immunology 5:647-656; Tuaillon et al. (1993) Proc. Nat'l. Acad. Sci. USA90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen et al.(1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol.152:2912-2920; Lonberg (1994) Handbook of Experimental Pharmacology113:49-101; Taylor et al. (1994) International Immunology 6: 579-591;Lonberg et al. (1995) Intern. Rev. Immunol. 13: 65-93; and Fishwild etal. (1996) Nature Biotechnology 14: 845-851; the contents of which arehereby incorporated by reference in their entireties. See also U.S. Pat.Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397;5,661,016; 5,814,318; 5,874,299; 5,770,429 and 5,545,807; andInternational Patent Application Publication Nos. WO 98/24884; WO94/25585; WO 93/1227; WO 92/22645 and WO 92/03918, the disclosures ofall of which are hereby incorporated by reference in their entireties.

To generate fully human monoclonal antibodies to IL-17A, HuMab mice areimmunized with an antigenic IL-17A polypeptide as described by Lonberget al. (1994); Fishwild et al. (1996) and WO 98/24884. Preferably, themice are 6-16 weeks of age upon the first immunization. For example, apurified preparation of IL-17A can be used to immunize the HuMab miceintraperitoneally. The mice can also be immunized with whole HEK293cells that are stably transformed or transfected with an IL-17A gene. An“antigenic IL-17A polypeptide” may refer to an IL-17A polypeptide of anyfragment thereof, which elicits an anti-IL-17A immune response in HuMabmice.

In general, HuMAb transgenic mice respond best when initially immunizedintraperitoneally (IP) with antigen in complete Freund's adjuvant,followed by every other week IP immunizations (usually up to a total of6) with antigen in incomplete Freund's adjuvant. Mice are immunizedfirst with cells expressing IL-17A (e.g., stably transformed HEK293cells), then with a soluble fragment of IL-17A, followed by alternatingimmunizations with the two antigens. The immune response is monitoredover the course of the immunization protocol with plasma samples beingobtained by retroorbital bleeds. The plasma are screened for thepresence of anti-IL-17A antibodies, for example by ELISA, and mice withsufficient titers of immunoglobulin are used for fusions. Mice areboosted intravenously with antigen three days before sacrifice andremoval of the spleen. Two to three fusions for each antigen may benecessary. Several mice are immunized for each antigen. For example, atotal of twelve HuMAb mice of the HCO7 and HCO12 strains can beimmunized.

Hybridoma cells producing the monoclonal, fully human anti-IL-17Aantibodies are produced by methods commonly known in the art, such asthe hybridoma technique originally developed by Kohler et al. (1975)(Nature 256:495-497); the trioma technique (Hering et al. (1988) Biomed.Biochim. Acta. 47:211-216 and Hagiwara et al. (1993) Hum. Antibod.Hybridomas 4:15); the human B-cell hybridoma technique (Kozbor et al.(1983) Immunology Today 4:72 and Cote et al. (1983) Proc. Nat'l. Acad.Sci. U.S.A. 80:2026-2030); and the EBV-hybridoma technique (Cole et al.(1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96). Preferably, mouse splenocytes are isolated and fused withPEG to a mouse myeloma cell line based on standard protocols. Theresulting hybridomas may then be screened for the production ofantigen-specific antibodies. In one embodiment, single cell suspensionsof splenic lymphocytes from immunized mice are fused to one-sixth thenumber of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL1580) with 50% PEG. Cells are plated at approximately 2×10⁵ cells/mL ina flat bottom microtiter plate, followed by a two week incubation inselective medium containing 20% fetal Clone Serum, 18% “653” conditionedmedia, 5% origen, 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES,0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/mlstreptomycin, 50 mg/ml gentamicin and 1×HAT (Sigma; the HAT is added 24hours after the fusion). After two weeks are cultured in medium in whichthe HAT is replaced with HT. Individual wells are then screened by ELISAfor human anti-IL-17A monoclonal IgG antibodies. Once extensivehybridoma growth occurs, medium is observed usually after 10-14 days.The antibody secreting hybridomas are replated, screened again, and ifstill positive for human IgG, anti-IL-17A monoclonal antibodies aresubcloned at least twice by limiting dilution. The stable subclones arethen cultured in vitro to generate small amounts of antibody in tissueculture medium for characterization.

In another embodiment, the anti-IL-17A antibody molecules of the presentinvention are produced recombinantly (e.g., in an E. coli/T7 expressionsystem). In this embodiment, nucleic acids encoding the antibodymolecules of the invention (e.g., V_(H) or V_(L)) are inserted into apET-based plasmid and expressed in the E. coli/T7 system. There areseveral methods to produce recombinant antibodies known in the art, e.g.U.S. Pat. No. 4,816,567 which is hereby incorporated by reference. Theantibody molecules may also be produced recombinantly in CHO or NSOcells.

Example 5 Indirect ELISA of Anti-IL-17A Monoclonal Antibodies

Binding of anti-human-IL-17A monoclonal antibodies to rhIL-17A isassessed using an indirect enzyme-linked immunosorbent assay (ELISA).Briefly, a fixed concentration of rhIL-17A is bound directly to thewells of a microtiter plate. The monoclonal anti-IL-17A to be assayed isthen is added to the rhIL-17A coated plate, where the antibody iscaptured and quantitated. A more detailed protocol follows.

A 96-well U-bottom MaxiSorp plate is coated with 50 μl/well of rhIL-17A(0.5 μg/ml) in carbonate coating buffer (the “assay plate”). Carbonatecoating buffer is 2.9 g/L NaHCO₃, 1.6 g/L Na₂CO₃, pH 9.4. Plates areincubated covered at 4° C. overnight. Monoclonal antibodies to bescreened are serially diluted in duplicate across the rows of a V-bottomplate such that the final volume is 60 μl/well (the “serial dilutionplate”). The assay plate is washed three times with PBS-Tween in a platewasher (SkanWasher, Molecular Devices, Sunnyvale, Calif., USA) andblotted dry. PBS-Tween is obtained by adding 0.5 ml/L Tween 20 to 1×PBS.Fifty μl from each well of the serial dilution plate is transferred tothe assay plate and incubated at 25° C. for one hour. Secondaryantibodies are diluted 1/2000 in diluent (PBS-BSA-Tween, which isPBS-Tween with 1 g/L BSA). The secondary antibody for rat monoclonalantibodies is goat anti-rat IgG (H+L)—HRP (Jackson ImmunoResearchLaboratories, Inc., West Grove, Pa., USA). The secondary antibody forchimeric and humanized monoclonal antibodies is F(ab′)₂ goat anti-humanIgG Fcγ—HRP (Jackson ImmunoResearch Laboratories, Inc.). The assay plateis washed as before. Diluted secondary antibodies (100 μl/well) areadded to the appropriate wells in the assay plate, and the plate isincubated at 25° C. for 45 minutes. The assay plate is washed as before.ABTS (100 μl/well) (Kirkegaard & Perry Laboratories, Gaithersburg, Md.,USA) is added, and the plate is incubated at 25° C. for 5-10 minutes,after which absorbance is read at 405 nm on a plate reader (Versamax,Molecular Devices, Sunnyvale, Calif., USA) with a 5 second shake beforereading.

Indirect ELISA results for various forms of antibody 16C10 of thepresent invention are shown in Table 5. Binding is reported as an EC50(the concentration of antibody necessary to obtain half-maximal signal).The results show that binding is detected with all forms of 16C10.Although such indirect ELISA assays are useful in quickly determiningthe presence or absence of anti-IL-17A antibodies, the EC50 numbersobtained may be assay-dependent and are typically not used to assess theabsolute binding affinity for any given antibody.

TABLE 5 Indirect Anti-IL-17A Antibody ELISA mAb rhIL-17A (μg) EC50 (pM)rat 16C10 0.025 274 chimeric 16C10 0.025 157 humanized 16C10 wt 0.025212

Example 6 ELISA of Anti-IL-17A Monoclonal Antibodies

Binding of anti-human-IL-17A monoclonal antibodies to rhIL-17A isassessed using an ELISA as follows. Briefly, a capture antibody is boundto the wells of a microtiter plate, after which a fixed concentration ofrhIL-17A is added. The monoclonal anti-IL-17A to be assayed is thentitrated versus the bound rhIL-17A on the plate to determine theconcentration of antibody needed to achieve half-maximal binding. A moredetailed protocol follows.

A 96-well microtiter plate is coated with 100 μl/well of captureantibody (rat anti-hIL-17A 12E6, 0.5 μg/ml) in carbonate coating bufferpH 9.5 (the “assay plate”). Plates are incubated covered at 4° C. for 24to 48 hours. The assay plate is washed three times in a plate washer(SkanWasher, Molecular Devices, Sunnyvale, Calif., USA) and blotted dry.The plate is then blocked with 200 μl/well of ELISA assay buffer (20 mMTris-HCl, 0.15 M NaCl, pH7.4, 0.5% BSA, 0.05% Tween-20, 2 mM EDTA) forone hour at 25° C. on an orbital shaker. The plate is washed, and 100Owen of either adenovirus-derived rhIL-17A or E. coli-derived humanIL-17 (IL-17A) (R&D Systems, Minneapolis, Minn., USA) (0.1 μg/ml) isadded in ELISA assay buffer and incubated for 2 hours at 25° C. on anorbital shaker. The plate is washed and the monoclonal antibodies to bescreened are serially diluted across a row of seven wells in the rangeof 1000 ng/ml to 0.0813 ng/ml using 4-fold serial dilutions. Plates areincubated for 1.5 hours at 25° C. on an orbital shaker. Plates arewashed and 100 μl/well secondary antibody (F(ab′)₂ goat anti-human kappalight chain—HRP, 1:20,000 dilution, BioSource, Carlsbad, Calif., USA) isadded, except for assay blank wells. Plates are washed twice (i.e. twocycles of 3 washes per cycle) with plate rotation between cycles. TMBsubstrate (Kirkegaard & Perry Laboratories, Gaithersburg, Md., USA) isadded at 100 μl/well and incubated 3-5 minutes on an orbital shaker.Stop solution is added (100 μl/well) and the plate is read forabsorbance at 450-570 nm on a plate reader (Versamax, Molecular Devices,Sunnyvale, Calif., USA).

ELISA results for various forms of antibody 16C10 of the presentinvention are shown in Table 6. Binding is reported as an EC50 (theconcentration of antibody necessary to obtain half-maximal signal). Theresults show that binding is detected with all forms of 16C10. Valuespresented with error ranges represent the mean of multipledeterminations with the standard deviation.

TABLE 6 Anti-IL-17A Antibody ELISA mAb human IL-17A EC50 (pM) hu 16C10wt rhIL-17A 66 ± 14 hu 16C10 wt R&D Systems 130 ± 19  hu 16C10 VH N54ArhIL-17A 75 hu 16C10 VH N54Q rhIL-17A 65 hu 16C10 VH N60A rhIL-17A 63 hu16C10 VH N60Q rhIL-17A 74 hu 16C10 VH M96L rhIL-17A 66 hu 16C10 VH M96ArhIL-17A 60 hu 16C10 VH M96K rhIL-17A 68 hu 16C10 VH M96F rhIL-17A 125hu 16C10 VH M100hF rhIL-17A 49 hu 16C10 VH M100hL rhIL-17A 53 hu 16C10(=VH N54Q/M96A) rhIL-17A 92 hu 16C10 (=VH N54Q/M96A) R&D Systems 136 hu16C10 VH 54Q/M96A/M100hF rhIL-17A 80 hu 16C10 VH 54Q/M96A/M100hF R&DSystems 118

Example 7 Binding Affinity of Anti-Human IL-17A Antibodies

Measuring Binding of Rat and Chimeric Anti-Human IL-17A Antibodies Usingan Electrochemiluminescence (ECL) Assay

Origen electrochemiluminescence technology, developed by IGEN, Inc.(Gaithersburg, Md., USA), and was employed to measure the binding of ratanti-human IL-17A antibodies (and one chimeric antibody) toFLAG-huIL-17A. See the Elecsys® immunoassay system, Roche Diagnostics(Indianapolis, Ind., USA). Electrochemiluminescence technology uses astable ruthenium metal chelate (Ori-TAG) which, in the presence oftripropylamine (TPA), generates electrochemiluminescence upon voltageapplication. Paramagnetic beads, microns in diameter, act as the solidphase and facilitate rapid assay kinetics. The bead/complex is channeledthrough a flow cell and captured at an electrode by magneticapplication. Voltage is applied and resulting electrochemiluminescenceis measured.

ECL assays were performed as follows. Three-fold serial dilutions ofanti-human IL-17A mAbs in 50 μl of the assay buffer were made in a96-well microtiter plate to give 1-3 μg/ml final concentration in thefirst well. Fifty μl of the assay buffer and 50 μl of biotinylatedFLAG-huIL-17A at 50 ng/ml was added to each well, followed by theaddition of either OriTag-labeled goat anti-rat IgG (H+L) pAb (50 μl at450 ng/ml) or an anti-hIgG mAb (50 μl at 500 ng/ml). Finally 50 μl ofOrigen Streptavidin-Dynabeads at 0.1 mg/ml was added to each well. After1 hr incubation at 25° C. the plate was processed by the Origen M-seriesM8/384 analyzer. GraphPad Prism software (GraphPad Software, San Diego,Calif., USA) was used to plot the data and calculate area under thecurve, which is a rough measure of binding.

Results are presented in Table 7 (which includes some duplicatedeterminations). The two rows showing binding of rat 16C10 toFLAG-huIL-17A represent duplicate determinations. All rat anti-humanIL-17A antibodies in the table (1D10, 16C10, 30C10, 23E12) bound toFLAG-huIL-17A, as did the chimeric 16C10. All four antibodies also boundto cyno IL-17A. Antibodies 16C10 and 30C10 did not bind to mouse IL-17Aunder the conditions of this assay, whereas antibodies 1D10 and 23E12did.

TABLE 7 Antibody Binding Determined by ECL Area Under mAb Antigen Peakrat 1D10 FLAG-hu-IL-17A 477474 rat 16C10 FLAG-hu-IL-17A 285792 rat 16C10FLAG-hu-IL-17A 374445 rat 30C10 FLAG-hu-IL-17A 311752 rat 23E12FLAG-hu-IL-17A 285145 chimeric 16C10 FLAG-hu-IL-17A 345982 rat 1D10 cynoIL-17 136497 rat 16C10 cyno IL-17 151543 rat 30C10 cyno IL-17 123916 rat23E12 cyno IL-17 111242 rat 1D10 mu IL-17 252121 rat 1D10 mu IL-17384999 rat 16C10 mu IL-17 no binding rat 16C10 mu IL-17 no binding rat30C10 mu IL-17 no binding rat 30C10 mu IL-17 no binding rat 23E12 muIL-17 143206 rat 23E12 mu IL-17 289185Determining the Equilibrium Dissociation Constant (K_(d)) for Rat andHumanized Anti-Human IL-17A Antibodies Using KinExA Technology

The equilibrium dissociation constants (K_(d)) for anti human IL-17Aantibodies were determined using the KinExA 3000 instrument (SapidyneInstruments Inc., Boise, Id., USA). KinExA uses the principle of theKinetic Exclusion Assay method based on measuring the concentration ofuncomplexed antibody in a mixture of antibody, antigen andantibody-antigen complex. See, e.g., Darling and Brault (2004) AssayDrug Dev. Technol. 2(6):647-57. The concentration of free antibody ismeasured by exposing the mixture to a solid-phase immobilized antigenfor a very brief period of time. In practice, this is accomplished byflowing the solution phase antigen-antibody mixture past antigen-coatedparticles trapped in a flow cell. Data generated by the instrument areanalyzed using custom software. Equilibrium constants are calculatedusing a mathematical theory based on the following assumptions:

1. The binding follows the reversible binding equation for equilibrium:k _(on)[Ab][Ag]=k _(off)[AbAg], where K _(d) =k _(off) /k _(on)

2. Antibody (Ab) and antigen (Ag) bind 1:1 and total antibody equalsantigen-antibody complex (AbAg) plus free antibody.

3. Instrument signal is linearly related to free antibody concentration.

KinExA analysis was performed on several rat anti-human IL-17Aantibodies, humanized variants thereof, and sequence variants of thesehumanized antibodies. IL-17A was derived from either human (“hu”),cynomolgus monkey (“cyno”), or mouse (“mu”). IL-17A from the samespecies was used in both the immobilized and solution phases for eachKinExA determination. Poly(methyl-methacrylate) (PMMA) particles (98micron) were coated with human, cyno or mouse IL-17A according toSapidyne “Protocol for coating PMMA particles with biotinylated ligandshaving short or nonexistent linker arms.” All experimental procedureswere done according to the KinExA 3000 manual. All runs were done induplicate.

The conditions for KinExA are provided at Table 8.

TABLE 8 KinExA Conditions IL-17A: human, cyno mouse Sample volume: 2 ml4 ml Sample flow rate: 0.25 ml/min 0.25 ml/min Label volume: 1 ml 1 mlLabel flow rate: 0.25 ml/min 0.25 ml/min Antibody conc.: 0.02-0.1 nM 0.1nM Highest antigen 4 nM 64 nM (23E12) conc.: 4.0 nM (1D10) Lowestantigen 1 pM 62 pM (23E12) conc.: 3.9 pM (1D10)

Two-fold serial dilutions of the antigen were prepared and mixed withthe antibody at constant concentration. The mixture was incubated for 2hours at 25° C. to equilibrate.

Table 9 shows the results of the KinExA analysis. Molar concentrationsfor the KinexA analysis were calculated on the basis of a molecularweight of 75 kDa for antibodies and 15 kDa for IL-17A to account for thepresence of two binding sites on the antibodies and the dimeric natureof IL-17A. For some antibodies, replicate experiments were performedwith different batches of antibody and/or antigen, in which cases meanvalues are provided in Table 9 along with standard errors. Bindingconstants for the humanized 16C10 wt antibody and the parental rat 16C10antibody were similar at approximately 5-10 pM, showing thathumanization did not significantly reduce the high affinity of theparental rat 16C10 for human IL-17A. Humanized 16C10 incorporatingvarious amino acid substitutions (N54Q, M96A, M100hF), including thefinal humanized 16C10 antibody (having N54Q and M96A substitutionscompared to rat 16C10) were also assayed and found to have similar, highbinding constants in the 1-10 pM range. The Fab fragment of hu 16C10bound retained high affinity (16 pM) compared with the completeantibody. Other antibodies of the present invention (rat 1D10, rat23E12, rat 30C10) also bound with high affinity to FLAG-huIL-17A, and tocyno IL-17A. Although rat 1D10 bound to mouse with 10 pM affinity,similar to its affinity for human and cyno IL-17A, rat 23E12 had200-2000 lower affinity for mouse IL-17A (7000 pM). Antibodies 16C10 and30C10 did not bind to mouse IL-17A (data not shown).

TABLE 9 K_(d) Values Determined by KinExA mAb Antigen Kd(pM) rat 16C10rhIL-17A 6.0 rat 16C10 FLAG-huIL-17A 3.6 hu 16C10 wt rhIL-17A 8.8 ± 3.0hu 16C10 [=VH N54Q/M96A] rhIL-17A 3.9 ± 2.7 hu 16C10 Fab rhIL-17A 16.1hu 16C10 VH N54A rhIL-17A 10.8 hu 16C10 VH N54Q rhIL-17A 7.0 hu 16C10 VHM96A rhIL-17A 9.9 hu 16C10 VH N54Q/M96A/M100hF rhIL-17A 10.0 rat 1D10FLAG-huIL-17A 1.7 rat 23E12 FLAG-huIL-17A 2.8 rat 30C10 FLAG-huIL-17A11.0 rat 1D10 cyno IL-17A 9.8 rat 23E12 cyno IL-17A 28.0 rat 30C10 cynoIL-17A 32.6 rat 16C10 cyno IL-17A 1.7 hu 16C10 cyno IL-17A 16.3 rat 1D10mu IL-17A 10.3 rat 23E12 mu IL-17A 7,000

Other methods known in the art, such as Biacore® surface plasmonresonance spectroscopy may be used to measure the affinity of antibodiesof the present invention. Although Biacore® analysis was performed onseveral of the antibodies of the present invention, the binding affinitywas generally too high to be measured accurately, specifically, thedissociation rate was too slow to be measured by this method. Suchanalysis may, however, be of use in the analysis of lower-affinityanti-IL-17A antibodies or anti-IL-17A antibodies having fasterdissociation rate constants.

Example 8 Synoviocyte Assay for Anti-IL-17A Antibodies

The ability of the anti-IL-17A antibodies of the present invention toblock the biological activity of IL-17A (either rhIL-17A or nativehuIL-17A) is measured by monitoring IL-17A-induced expression of IL-6and IL-8 in primary culture of human synoviocytes, as follows.Synoviocytes are isolated by collagenase digestion of a rheumatoidarthritis synovium obtained from a knee replacement patient.Synoviocytes are enriched by continuous passage in Growth Medium (DMEM,10% BCS, 1× Pen-Strep (50 IU/ml penicillin, 55 μg/ml streptomycin), 1×beta-mercaptoethanol (50 μM), 1× glutamine (20 mM), 25 mM HEPES), frozendown at passage number three, and stored in liquid nitrogen. When readyfor use in an assay, a vial of the cells is thawed, plated, and thecells are allowed to grow to near confluence. The cells are thenpassaged 1:2 into larger flasks using typsin/EDTA. When sufficient cellshave expanded, an experiment is initiated by trypsinizing the cells,plating into 96 well or 48 well plates, and allowing them to grow tototal confluence.

IL-17A is diluted to 120 ng/ml, i.e. 4× the final concentration of 30ng/ml (1 nM). IL-17A is either human (rhIL-17A and native huIL-17A) orfrom a non-human primate, in this case cynomolgus monkeys (cyno). 100and 300 μl aliquots of the 4×IL-17A stocks are added to empty 96-welland 48-well plates, respectively.

Anti-IL-17A antibodies to be assayed are diluted in Growth Medium to 4×the maximum concentration to be tested in an experiment. The 4×anti-IL-17A stock is serially diluted 1:2 to cover the dynamicinhibition range of the assay. Each of the serially diluted antibodysamples (all are 4× their final concentration) are mixed 1:1 with the4×IL-17A solutions in empty plates to generate mixtures with 2×concentrations of both IL-17A and anti-IL-17A antibodies. These mixturesare allowed to equilibrate at 37° C. in a tissue culture incubator formore than four hours.

Medium is removed from the adherent confluent synoviocytes and replacedwith 100 μl (96-well plate) or 200 μl (48-well plate) of Growth Medium.An equal volume of the 2× ligand/2× antibody solution is added tosynoviocytes to give 1×IL-17A (30 ng/ml final) and 1× antibody. Eachwell (data point) is run in duplicate. Synoviocytes are activated (i.e.exposed to the IL-17A/antibody mixture) for three days, at which pointsupernatants are transferred to 96 well plates, and optionally frozen,and stored at −80° C. until analyzed. Microtiter plates containingsupernatants are thawed and each solution is diluted 1:10 using GrowthMedium. Supernatants are analyzed for IL-6 and IL-8 using Luminex beadpairs (Upstate, Charlottesville, Va., USA) following manufacturer'sinstructions.

Results are provided at Tables 10 (IL-6) and 11 (IL-8). Values presentedwith error ranges represent the mean of multiple determinations with thestandard deviation. Results for various forms of antibody 16C10 areshown, including a humanized form of 16C10 having the original rat CDRs(“hu16C10 (wt)”) as well as several variants having one, two or threechanges in the heavy chain CDRs (generally “hu16C10 X##Z”, where X isthe amino acid at residue ## in the heavy chain of hu16C10 (wt) and Z isthe new amino acid). “NHP IL-17A” is non-human primate-derived IL-17A,in this case cynomolgus monkey IL-17A. “Native huIL-17A” refers tomature huIL-17A produced when the precursor protein is produced usingthe natural signal sequence, and differs from rhIL-17A by the absence oftwo N-terminal amino acids. Concentrations and IC50 values are expressedin ng/ml, but may be expressed in pM units as well. For example, 30ng/ml rhIL-17A corresponds to 1000 pM (MW=30 kDa) and 70 ng/mlanti-IL-17A antibody corresponds to approximately 470 pM (MW=150 kDa).

TABLE 10 IC50 (ng/ml) of Anti-IL-17A Measured by Synoviocyte IL-6Production rhIL-17A NHP IL-17A Native huIL-17A Antibody (30 ng/ml) (30ng/ml) (10 ng/ml) rat 1D10 105 ± 28 65 ± 15 25 rat 16C10 63 ± 7 60 ± 10hu16C10 (wt)  80 ± 10 — hu16C10 (N54A) 60 — hu16C10 (N54Q) 60 — hu16C10(M96A) 60 — hu16C10 (M96K) 60 — hu16C10 (M100hF) 70 — hu16C10 70 ± 8 70± 0  25 (N54Q/M96A) hu16C10 70 — (N54Q/M96A/M100hF)

TABLE 11 IC50 (ng/ml) of Anti-IL-17A Measured by Synoviocyte IL-8Production rhIL-17A NHP IL-17A Native huIL-17A Antibody (30 ng/ml) (30ng/ml) (10 ng/ml) 1D10 59 ± 41 38 ± 13 40 16C10 38 ± 14 33 ± 8  hu16C10(wt) 42 ± 16 — hu16C10 (N54A) 25 — hu16C10 (N54Q) 25 — hu16C10 (M96A) 25— hu16C10 (M96K) 25 — hu16C10 (M100hF) 50 — hu16C10 38 ± 10 38 ± 13 50(N54Q/M96A) hu16C10 40 — (N54Q/M96A/M100hF)

Example 9 NHDF Assay for Anti-IL-17A Antibodies

The ability of the anti-IL-17A antibodies of the present invention toblock the biological activity of IL-17A is measured by monitoringrhIL-17A-induced expression of IL-6 in a normal human (adult) dermalfibroblast (NHDF) primary cell line. Briefly, various concentrations ofan anti-IL-17A antibody to be assayed are incubated with rhIL-17A, andthe resulting mixture is then added to cultures of NHDF cells. IL-6production is determined thereafter as a measure of the ability of theantibody in question to inhibit IL-17A activity. A more detailedprotocol follows.

A series two-fold dilutions of anti-IL-17A antibodies of interest areprepared (in duplicate) starting with a stock solution at 40 μg/ml. Astock solution of rhIL-17A is prepared at 120 ng/ml. Seventy μl of therhIL-17A stock solution is mixed with 70 μl of the anti-IL-17A antibodydilutions in wells of a microtiter plate and incubated at roomtemperature for 20 minutes. One hundred μl of each of these mixtures isthen added to wells of a microtiter plate that had been seeded with1×10⁴ NHDF cells/well (100 μl) the previous night and allowed toincubate at 37° C. NHDF cells (passage 4) were obtained from Cambrex BioScience (Baltimore, Md., USA). The resulting final concentration ofrhIL-17A is 30 ng/ml (1 nM), and the antibodies range downward intwo-fold intervals from 10 μg/ml. Plates are incubated at 37° C. for 24hours, followed by harvesting of the supernatant and removal of 50 μlfor use in an IL-6 ELISA.

The ELISA for detection of human IL-6 is performed as follows. Reagentsare generally from R&D Systems (Minneapolis, Minn., USA). An hIL-6capture antibody (50 μl/well of a 4 μg/ml solution) is transferred towells of a microtiter plate, which is sealed and incubated overnight at4° C. The plate is washed three times, and then blocked with 100 μl/wellof blocking buffer for 1 hour or more The plate is then washed againthree times. Experimental samples (50 μl of the culture supernatant) andcontrols (serial dilutions of IL-6 protein) are added to the wells in 50μl and incubated for two hours. Plates are washed three times, and 50μl/well of a biotinylated anti-IL-6 detection antibody (300 ng/ml) isadded. The plates are incubated at room temperature for two hours,washed three times, and 100 μl/well of streptavidin HRP is added andincubated for 20 minutes. The plate is washed again, ABTS (BioSource,Carlsbad, Calif., USA) is added (100 μl/well), and incubated for 20minutes. Stop solution is added (100 μl/well) and the absorbance at 405nm is measured.

The IC50 for an anti-IL-17A antibody of interest is the concentration ofantibody required to reduce the level of rhIL-17A-induced IL-6production to 50% of the level observed in the absence of any addedanti-IL-17A antibody.

Results are provided at Table 12.

TABLE 12 Anti-IL-17A Antibody Inhibition of IL-6 Production in NHDFCells rhIL-17A cyno IL-17A Antibody IC50 (nM) IC50 (nM) rat 4C3 0.5 0.2rat 16C10 0.5 0.2 rat 30C10 0.5 0.2 rat 6C3 0.8 0.2 rat 1D10 1 0.4 rat8G9 1 0.4 rat 12B12 1 0.4 rat 18H6 1 0.3 23E12 1 0.3 29G3 1.5 2 29H1 1.50.5 12E6 >70 >70

Example 10 Foreskin Fibroblast Assay Anti-IL-17A Antibodies

The ability of the anti-IL-17A antibodies of the present invention toblock the biological activity of IL-17A is measured by monitoringrhIL-17A-induced expression of IL-6 in HS68 foreskin fibroblast cellline. Reduced production of IL-6 in response to rhIL-17A is used as ameasure of blocking activity by anti-IL-17A antibodies of the presentinvention.

Analysis of the expression of IL-17RC (an IL-17A receptor) in a panel offibroblast cell lines identified the human foreskin fibroblast cell lineHS68 (ATCC CRL1635) as a potential IL-17A responsive cell line. This wasconfirmed by indirect immunofluorescence staining with polyclonal goatanti-human IL-17R antibody (R&D Systems, Gaithersburg, Md., USA)followed by phycoerythrin (PE)-F(ab′)₂ donkey anti-goat IgG (JacksonImmunoresearch, Inc., West Grove, Pa., USA), and analyzing the PEimmunofluorescence signal on a flow cytometer (FACScan,Becton-Dickinson, Franklin Lakes, N.J., USA). As further validation ofthe model, IL-17A (both adenovirus-derived rhIL-17A and commerciallyavailable E. coli-derived IL-17A, R&D Systems) induced a dose-responsiveinduction of IL-6 in the HS68 cells with an EC50 of 5-10 ng/ml, whichinduction was blocked by pre-incubation with commercial polyclonal andmonoclonal anti-IL-17A antibodies (R&D Systems).

The IL-17A inhibition assay is performed as follows. A confluent T-75flask of HS68 cells (approximately 2×10⁶ cells) is washed withDulbecco's PBS without Ca++ and Mg++ and then incubated with 5 ml ofcell dissociation medium (Sigma-Aldrich, St. Louis, Mo., USA) for 2-5minutes at 37° C. in an incubator at 5% CO₂. Cells are then harvestedwith 5 ml of tissue culture (TC) medium and centrifuged for 5 minutes at1000 rpm. TC medium is Dulbecco's Modified Eagle's Medium (withglutamine), 10% heat-inactivated fetal bovine serum (Hyclone), 10 mMHepes, 1 mM sodium pyruvate, penicillin, and streptomycin. Cells areresuspended in 2 ml TC medium, diluted 1:1 with trypan blue and counted.Cell concentrations are adjusted to 1×10⁵ cells/ml in TC medium, and 0.1ml/well is aliquoted into the wells of a flat-bottom plate containing0.1 ml TC medium. Cells are grown overnight and the supernatant isaspirated and cells are washed with 0.2 ml of fresh TC medium.

Anti-IL-17A antibodies to be assayed are serially diluted in two-fold or3-fold steps to give a series of stock solutions that can be used tocreate final antibody concentrations of 1 to 0.001 μg/ml in the IL-17Ainhibition assay. A rat IgG control is used in each assay, as well asmedia-only samples, as controls to measure spontaneous IL-6 productionin HS68 cells. The TC medium is aspirated from the wells of the platecontaining the HS68 cells. Aliquots of the various concentrations ofanti-IL-17A antibody (0.1 ml of each) are pre-incubated in the wellswith the HS68 cells 37° C. for 5 minutes prior to addition of 0.1 ml of20 ng/ml rhIL-17A, to give a final concentration of rhIL-17A of 10 ng/ml(approximately 330 pM of IL-17A dimer). Cells are incubated 24 hours at37° C., and supernatants (50-100 μl) are harvested and assayed for IL-6,for example using a human IL-6 ELISA kit from Pharmingen (OptEIA—BDBiosciences, Franklin Lakes, N.J., USA).

Results for several rat anti-human IL-17A antibodies of the presentinvention in the foreskin fibroblast IL-17A inhibition assay areprovided at Table 13.

TABLE 13 Foreskin Fibroblast Assay anti-IL-17A antibody IC50 (pM) rat16C10 67 rat 1D10 65 rat 8G9 29 rat 29H1 247 rat 29G3 63 rat 23E12 126rat 6C3 192 rat 4C2 107 rat IgG1 no binding

Example 11 Ba/F3-hIL-17Rc-mGCSFR Proliferation Assay

The ability of the anti-IL-17A antibodies of the present invention toblock the biological activity of IL-17A is measured by monitoringrhIL-17A-induced proliferation of a cell line engineered to proliferatein response to IL-17A stimulation. Specifically, the Ba/F3 cell line(IL-3 dependent murine pro-B cells) was modified to express a fusionprotein comprising the extracellular domain of a human IL-17A receptor(hIL-17RC) fused to the transmembrane domain and cytoplasmic region ofmouse granulocyte colony-stimulating factor receptor (GCSFR). Theresulting cell line is referred to herein as Ba/F3 hIL-17Rc-mGCSFR.Binding of homodimeric IL-17A to the extracellular IL-17RC domainscauses dimerization of the hIL-17Rc-mGCFR fusion protein receptor, whichsignals proliferation of the Ba/F3 cells via their mGCSFR cytoplasmicdomains. Such cells proliferate in response to IL-17A, providing aconvenient assay for IL-17A inhibitors, such as anti-IL-17A antibodies.

The sensitivity of the Ba/F3-hIL-17Rc-mGCSFR proliferation assay toIL-17A stimulation makes it possible to perform experiments atrelatively low concentrations of rhIL-17A (e.g. 3 ng/ml, 100 pM)compared with other assays, while still maintaining a robust and readilymeasurable proliferative response. This means that lower concentrationsof anti-IL-17A antibodies are required to achieve a molar excess overrhIL-17A in the assay. Experiments performed at lower antibodyconcentrations make it possible to discriminate between high affinityantibodies that might otherwise be indistinguishable (i.e. experimentscan be performed closer to the linear range in the antibody-IL-17Abinding curve, rather than in the plateau).

Antibodies and IL-17A were filtered through 0.22 μm filters afterdilution to working stock concentrations but prior to addition toexperimental samples. Four sets of samples were prepared, in duplicate,across rows of a 96-well flat bottom tissue culture plates. As used inthis Example, Growth Medium is RPMI 1640 w/Glutamax (Invitrogen,Carlsbad, Calif., USA), 55 μM 2-mercaptoethanol, 10% formula fed BovineCalf Serum (Irvine Scientific, Santa Ana, Calif., USA), 50 μg/mLgentamicin, 2 μg/mL puromycin, and 10 ng/mL mIL-3 BioAssay Medium is thesame as Growth Medium but without puromycin and mIL-3. All serialdilutions in this Example were made into BioAssay Medium.

The following experimental samples (75 μl) were prepared: 1) a serialdilution of Growth Medium (including 10 ng/ml mIL-3); 2) a serialdilution of rhIL-17A; 3) a serial dilution of anti-IL-17A antibodies ofthe present invention mixed with 3 ng/ml IL-17A (final concentrationafter cells were added), including a “no antibody” control; and 4) a“cells only” control with no added antibodies, IL-17A or mIL-3. Ba/F3hIL-17Rc-mGCSFR cells (7500 cells/well) were then added to bring thetotal volume to 100 μl/well, and the plates were incubated at 37° C./5%CO₂ for approximately 40 hours. AlamarBlue® indicator dye (11 μl/well)was added and the plates were incubated at 37° C./5% CO₂ for 6-8 hours.Plates were then read for the difference in absorbance at 570 nm and 600nm. IC50 values were determined using nonlinear fit/sigmoidaldose-response/variable slope.

The results of the Ba/F3 hIL-17Rc-mGCSFR proliferation assay areprovided at Table 14.

TABLE 14 Ba/F3 hIL-17Rc-mGCSFR Proliferation Assay mAb IL-17A[rhIL-17A](pM) IC50(pM) rat 16C10 human 100 20 ± 8 rat 16C10 human 276162 rat 16C10 cyno 100 27 chimeric 16C10 human 100 29 hu 16C10 human 10015 ± 2 hu 16C10 human 100 13 VH N54Q/M96A hu 16C10 human 100 27 hu 16C10human 276 149 hu 16C10 cyno 100 17 hu 16C10 N54Q/M96A/M100hF human 10011 rat 1D10 human 276 223 rat 1D10 cyno 100 19 rat 29H1 human 28 ≧30 rat4H12 human 28 ≧400 rat 29G3 human 28 ≧400

Example 12 Cross-Blocking of Anti-IL-17A Antibodies

Different anti-IL-17A antibodies of the invention may bind to the sameepitope, epitopes that overlap, or epitopes that do not overlap,including epitopes that are sufficiently distinct that two or moreantibodies can bind to one IL-17A monomer simultaneously. Antibodiesthat bind to portions of IL-17A critical to receptor binding will blockthe receptor-mediated biological activity of IL-17A. Such antibodies arereferred to herein as “neutralizing antibodies.” Antibodies that bindbut do not block receptor binding are referred to as non-neutralizingantibodies.

When performing experiments on IL-17A and anti-IL-17A antibodies it isuseful to be able to determine the level of IL-17A (or anti-IL-17A) in asample, such as by a sandwich ELISA. See, e.g., Example 6. In oneformat, an IL-17A ELISA involves coating the wells of a microtiter platewith a capture antibody, addition of an experimental sample possiblycontaining IL-17A, and binding of a detection antibody. The captureantibody and the detection antibody must be able to bind IL-17A at thesame time.

A similar assay may be used to determine the level of an anti-IL-17Aantibody, wherein a standard solution of IL-17A is bound to the wellscoated with capture antibody, followed by addition of a an experimentalsample possibly containing an anti-IL-17A antibody, and binding of asecondary detection antibody (e.g. an anti-human IgG antibody in thecase of IgG humanized antibodies of the present invention). As in theIL-17A sandwich ELISA, the capture antibody cannot interfere withbinding of the antibody to be assayed.

Preferred pairs of antibodies for use in the ELISA experiments outlinedin this Example can be determined by performing cross-blockingexperiments. In cross blocking experiments, a first antibody is coatedonto the wells of a microtiter plate. A biotinylated second antibody isthen mixed with IL-17A and allowed to bind, after which the mixture isadded to the coated well and incubated. The biotinylated second antibodymay be added at various concentrations (i.e. titrated) to ensure that inat least some samples the antibody is present at a two-fold (or greater)molar excess over homodimeric IL-17A. The plate is then washed and thepresence or absence of the biotinylated second antibody bound in thewell is determined by standard methods.

If the two antibodies cross-block there will be a reduction of signal(IL-17A binding) to the plate in the presence of the second anti-IL-17Aantibody compared with control samples containing no second anti-IL-17Aantibody (or containing an isotype control). Pairs of antibodies that donot cross-block can be used together in assays, such as sandwich ELISAs.Although the dimeric nature of IL-17A makes it possible to use pairs ofcross-blocking antibodies in ELISAs in certain formats (e.g. whereIL-17A is bound to the capture antibody on the plate prior to additionof the detection antibody), non-cross-blocking pairs of antibodies aregenerally preferable.

Several anti-IL-17A antibodies of the present invention (clones 4C3,6C3, 8G9, 12E6, 16C10, 18H6, 23E12, 29H1, 30C10, 1D10, 21B12, 29G3) weretested pairwise for cross-blocking. All pairs cross-blocked with theexception of 29G3/1D10 and 29G3/21B12, which pairs of antibodies couldtherefore be used in ELISAs. In addition to identifying pairs ofanti-IL-17A antibodies that can be used in an ELISA, these results showthat the epitope bound by antibody 29G3 is functionally or physicallydistinct from the epitope or epitopes bound by antibodies 1D10 and21B12. These data also demonstrated that the epitopes for 1D10 and 21B12overlap with, but are not identical to, the epitope for 16C10.

Such pairs of anti-IL-17A antibodies that bind to functionally distinctepitopes are useful, e.g., in validating anti-IL-17Aimmunohistochemistry (IHC). For example, if a tissue sample exhibits thesame pattern of IL-17A expression in IHC performed with two differentanti-IL-17A antibodies that bind to functionally distinct epitopes thenit is even more likely that the assay is detecting IL-17A, rather thansome other spurious cross-reacting protein in the tissue sample.

Such pairs of non-cross-blocking antibodies are also useful in designingELISAs for detection of IL-17A in the presence of therapeuticanti-IL-17A antibodies, e.g. in samples from patients undergoinganti-IL-17A antibody therapy, in which the presence of an excess of thetherapeutic anti-IL-17A antibody would block detection by anti-IL-17AELISA unless the ELISA antibodies were non-cross-blocking with thetherapeutic antibody.

Example 13 Gene Therapy with Anti-IL-17A Antibodies

The anti-IL-17A antibodies of the invention may also be administered toa subject by gene therapy. In a gene therapy approach, the cells of asubject are transformed with nucleic acids that encode the antibodies ofthe invention. Subjects comprising the nucleic acids will then producethe antibody molecules (intrabodies) endogenously. For example, Alvarezet al. introduced single-chain anti-ErbB2 antibodies to subjects using agene therapy approach. Alvarez et al. (2000) Clinical Cancer Research6:3081-3087. The methods disclosed by Alvarez et al. may be easilyadapted for the introduction of nucleic acids encoding an anti-IL-17Aantibody molecule of the present invention to a subject. In oneembodiment, the antibody molecule introduced by gene therapy is a fullyhuman, single-chain antibody.

The gene therapy approach described herein has the potential advantagethat treatment need only be carried out once, or at most a limitednumber of times, provided that long-term gene expression is achieved.This is contrasted with antibody administration, which must be repeatedperiodically to maintain proper therapeutic levels in the subject.

The nucleic acids may be introduced to the cells of a subject by anymeans known in the art. In some embodiments, the nucleic acids areintroduced as part of a viral vector. Examples of viruses from which thevectors may be derived include lentiviruses, herpes viruses,adenoviruses, adeno-associated viruses (AAV), vaccinia virus,baculovirus, alphavirus, influenza virus, and other recombinant viruseswith desirable cellular tropism. Various companies produce viral vectorscommercially, for example Avigen, Inc. (Alameda, Calif.; AAV vectors);Cell Genesys (Foster City, Calif.; retroviral, adenoviral, AAV vectors,and lentiviral vectors); Clontech (retroviral and baculoviral vectors);Genovo, Inc. (Sharon Hill, Pa.; adenoviral and AAV vectors); Genvec(adenoviral vectors); IntroGene (Leiden, Netherlands; adenoviralvectors); Molecular Medicine (retroviral, adenoviral, AAV, and herpesviral vectors); Norgen (adenoviral vectors); Oxford BioMedica (Oxford,United Kingdom; lentiviral vectors); and Transgene (Strasbourg, France;adenoviral, vaccinia, retroviral, and lentiviral vectors).

Methods of constructing and using viral vectors are known in the art(see, e.g., Miller et al. (1992) BioTechniques 7:980-990). Preferably,the viral vectors are replication defective (unable to replicateautonomously) and thus not infectious in the target cell. Preferably,the replication defective virus is a minimal virus retaining only thesequences of its genome that are necessary for encapsidating the genometo produce viral particles. Defective viruses that entirely or almostentirely lack viral genes are most preferred. Use of defective viralvectors allows for administration to cells in a specific localized areawithout concern that the vector can infect other cells, enablingtissue-specific targeting. See, e.g., Kanno et al. (1999) Cancer Gen.Ther. 6:147-154; Kaplitt et al. (1997) J. Neurosci. Meth. 71:125-132;and Kaplitt et al. (1994) J. Neuro-Onc. 19:137-142.

Adenoviruses are eukaryotic DNA viruses that can be modified toefficiently deliver a nucleic acid of the invention to a variety of celltypes. Attenuated adenovirus vectors, such as the vector described byStratford-Perricaudet et al. (1992) (J. Clin. Invest. 90:626-630) aredesirable in some instances. Various replication defective adenovirusand minimal adenovirus vectors have been described (PCT Publication Nos.WO94/26914, WO94/28938, WO94/28152, WO94/12649, WO95/02697 andWO96/22378). The replication defective recombinant adenoviruses of thepresent invention can be prepared by any technique known to a personskilled in the art (see Levrero et al. (1991) Gene 101:195; EP 185573;Graham (1984) EMBO J. 3:2917; Graham et al. (1977) J. Gen. Virol.36:59).

Adeno-associated viruses (AAV) are DNA viruses of relatively small sizethat can integrate, in a stable and site-specific manner, into thegenome of the cells that they infect. They are able to infect a widespectrum of cells without inducing any effects on cellular growth,morphology or differentiation, and they do not appear to be involved inhuman pathologies. The use of AAV-derived vectors for transferring genesin vitro and in vivo has been described (see Donsante et al. (2001) GeneTher. 8:1343-1346; Larson et al. (2001) Adv. Exp. Med. Bio. 489:45-57;PCT Publication Nos. WO91/18088 and WO93/09239; U.S. Pat. Nos. 4,797,368and 5,139,941; and EP 488528B1).

In another embodiment, the gene can be introduced in a retroviralvector, e.g., as described in U.S. Pat. Nos. 5,399,346, 4,650,764,4,980,289, and 5,124,263; Mann et al. (1983) Cell 33:153; Markowitz etal. (1988) J. Virol. 62:1120; EP 453242 and EP178220. Retroviruses areintegrating viruses that infect dividing cells.

Lentiviral vectors can be used as agents for the direct delivery andsustained expression of nucleic acids encoding an antibody molecule ofthe invention in several tissue types, including brain, retina, muscle,liver and blood. The vectors can efficiently transduce dividing andnondividing cells in these tissues, and maintain long-term expression ofthe antibody molecule. For a review, see Zufferey et al. (1998) J.Virol. 72:9873-80 and Kafri et al. (2001) Curr. Opin. Mol. Ther.3:316-326. Lentiviral packaging cell lines are available and knowngenerally in the art, facilitating the production of high-titerlentivirus vectors for gene therapy. An example is atetracycline-inducible VSV-G pseudotyped lentivirus packaging cell linewhich can generate virus particles at titers greater than 10⁶ IU/ml forat least 3 to 4 days; see Kafri et al. (1999) J. Virol. 73: 576-584. Thevector produced by the inducible cell line can be concentrated as neededfor efficiently transducing nondividing cells in vitro and in vivo.

Sindbis virus is a member of the alphavirus genus that has been studiedextensively since its discovery in various parts of the world beginningin 1953. Gene transduction based on alphavirus, particularly Sindbisvirus, has been well-studied in vitro (see Straus et al. (1994)Microbiol. Rev. 58:491-562; Bredenbeek et al. (1993) J. Virol. 67;6439-6446; Iijima et al. (1999) Int. J. Cancer 80:110-118; and Sawai etal. (1998) Biochim. Biophys. Res. Comm. 248:315-323). Many properties ofalphavirus vectors make them a desirable alternative to othervirus-derived vector systems being developed, including rapidengineering of expression constructs, production of high-titered stocksof infectious particles, infection of nondividing cells, and high levelsof expression (Strauss et al. (1994) Microbiol. Rev. 58:491-562). Use ofSindbis virus for gene therapy has been described. (Wahlfors et al.(2000) Gene. Ther. 7:472-480 and Lundstrom (1999) J. Recep. Sig.Transduct. Res. 19(1-4):673-686).

In another embodiment, a vector can be introduced to cells bylipofection or with other transfection facilitating agents (peptides,polymers, etc.). Synthetic cationic lipids can be used to prepareliposomes for in vivo and in vitro transfection of a gene encoding amarker (Felgner et al. (1987) Proc. Nat'l. Acad. Sci. USA 84:7413-7417and Wang et al. (1987) Proc. Nat'l. Acad. Sci. USA 84:7851-7855). Usefullipid compounds and compositions for transfer of nucleic acids aredescribed in PCT Publication Nos. WO 95/18863 and WO96/17823, and inU.S. Pat. No. 5,459,127.

It is also possible to introduce the vector in vivo as a naked DNAplasmid. Naked DNA vectors for gene therapy can be introduced into thedesired host cells by methods known in the art, e.g., electroporation,microinjection, cell fusion, DEAE dextran, calcium phosphateprecipitation, use of a gene gun, or use of a DNA vector transporter(see, e.g., Wilson et al. (1992) J. Biol. Chem. 267:963-967; Williams etal. (1991) Proc. Nat'l. Acad. Sci. USA 88:2726-2730). Receptor-mediatedDNA delivery approaches can also be used (Wu et al. (1988) J. Biol.Chem. 263:14621-14624). U.S. Pat. Nos. 5,580,859 and 5,589,466 disclosedelivery of exogenous DNA sequences, free of transfection facilitatingagents, in a mammal. A relatively low voltage, high efficiency in vivoDNA transfer technique, termed electrotransfer, has also been described(Vilquin et al. (2001) Gene Ther. 8:1097; Payen et al. (2001) Exp.Hematol. 29:295-300; Mir (2001) Bioelectrochemistry 53:1-10; PCTPublication Nos. WO99/01157, WO99/01158 and WO99/01175).

The gene therapy methods outlined herein may be carried out in vivo, orthey may be performed ex vivo, in which cells are obtained from asubject, transformed with a gene therapy method in vitro, andsubsequently reintroduced into the subject. See, e.g., Worgall (2005)Pediatr. Nephrol. 20(2):118-24.

Example 14 Cassette Mutagenesis of CDRs of Parental Antibodies

Optimization of the CDR sequences of anti-human IL-17A antibodies of thepresent invention (e.g. 16C10) is performed using shotgun scanningmutagenesis. Alanine scanning mutagenesis is used to determine whichresidues within the CDRs are most critical to IL-17A binding (seeExample 19). The codon for one or more residues within one or more CDRsis replaced with an alanine codon, or an alanine codon is replace with aglycine codon, and the resulting antibody is tested for a relevantactivity (e.g. IL-17A binding affinity, IC50 for receptor blocking in acompetition assay, bioassay, as provided in various other Examplesherein). Codon substitution may be performed by any method known in theart, including but not limited to site-directed mutagenesis (e.g.Kunkel, Proc. Nat'l. Acad. Sci. USA (1985) 82:488) and PCR mutagenesis.Residues crucial to IL-17A binding may also be determined by inspectionof the structure of an IL-17A-antibody complex, e.g. an X-ray crystalstructure. Antibody CDR residues within contact distance of IL-17A, orwhich are substantially buried in formation of the IL-17A-antibodycomplex, are candidates for further optimization.

Those residues with the greatest sensitivity to mutation are thenstudied further, for example by homolog scanning mutagenesis. In thisembodiment, conservative amino acid substitutions with homologous aminoacids are performed at the target residues to search for antibodies withsuperior qualities. Non-conservative mutations are also possible, albeitat the risk of disrupting IL-17A binding altogether.

Alternatively, improved antibody sequences may be generated usingaffinity maturation, in which selected residues in a CDR are mutated togenerate all possible amino acid substitutions at that position. Inanother embodiment, fewer than all 20 possible natural amino acids areused as substitutions to reduce the number of potential sequences tomore manageable levels, while still providing for chemical diversity ateach position using a limited number of amino acids selected to beoptimally diverse (e.g. representative hydrophobic, polar-uncharged,basic and acidic amino acids), as in WO2005/044853. Such affinitymaturation can be performed by substitution with any number of aminoacids at a position of interest, including 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or even more if non-standard ormodified amino acids are included.

Example 15 Cross-Reactivity with Human Tissue

The propensity of humanized anti-human IL-17A antibody hu16C10 tocross-reactivity with non-target tissues in human subjects was assessedas follows. hu16C10 was preincubated with a biotinylated secondaryantibody to form a pre-complex prior to exposure to tissue samples.Antibody complexes (with 20 μg/ml of antibody) were then mixed with atissue or other sample and incubated to allow binding. Bound secondaryantibodies were then detected using ABC immunoperoxidase detection(Vector Labs, Burlingame, Calif., USA). Tuson et al. (1990) J. HistochemCytochem. 38(7):923-6. A sample with an unrelated human IgG1 antibodywas included as a negative control.

Immunohistochemical (IHC) staining was performed with hu16C10 againstseveral positive control target tissues including rhIL-17A protein spotson UV-resin slides (Adhesive Coated Slides, Instrumedics, Inc., St.Louis, Mo., USA), mouse liver cells infected with an adenovirus-encodingIL-17A, and human rheumatoid arthritis tissue. IHC revealed binding(+++) in all three positive controls.

IHC was then performed against a panel of human tissues (32 in all) toassess cross-reactivity. All these human tissue samples were mounted onUV-resin slides. Samples were obtained from three donors for eachtissue. The human tissues screened were adrenal gland, bladder,cerebellum, cerebral cortex, colon, fallopian tube, cardiac muscle,kidney, liver, lung, lymph node, mammary gland, ovary, pancreas,parathyroid, pituitary gland, placenta, prostate, retina, skeletalmuscle, skin, small intestine, spinal cord, spleen, stomach, testis,thymus, thyroid gland, ureter, uterus, and cervix (uterus). IHC wasnegative in all 32 tissues.

This lack of cross-reactivity has several potential benefits intherapeutic uses of the anti-IL-17A antibodies of the present invention,such as reducing the loss of antibody due to non-specific binding toother tissues (with consequent reduction in therapeutic effect), andreducing the likelihood of adverse effects associated with binding toundesired tissues.

Example 16 Treatment of Collagen-Induced Arthritis Using Anti-IL-17AAntibodies

Collagen-induced arthritis (CIA) is a widely accepted mouse model forrheumatoid arthritis in humans. Anti-IL-17A antibody 1D10 of the presentinvention (the parental rat antibody, rather than a humanized formsthereof) is administered to mice expressing CIA to assess the ability ofanti-IL-17A therapy to treat rheumatoid arthritis.

The procedure was as follows. On Day 0 male B10.RIII mice were immunizedintradermally at the tail base with bovine type II collagen emulsifiedin Complete Freund's Adjuvant. On Day 21 mice were challengedintradermally with bovine type II collagen emulsified in IncompleteFreund's Adjuvant delivered at the tail base. When the first sign ofsevere arthritis in the immunized group occurred (post-Day 21), allremaining immunized mice were randomized to the various treatmentgroups. Animals were treated with either 800 μg, 200 μg, or 50 μg ofanti-IL-17A antibody 1D10; 200 μg isotype control antibody; or diluent.Treatments were given subcutaneously on the first day of disease onsetin the immunized mice, and then weekly four more times. Mice weresacrificed at day 35 and paws were fixed in 10% neutral-bufferedformalin for tissue processing and sectioning. Paws were analyzed by apathologist for the following histopathology parameters: reactivesynovium, inflammation, pannus formation, cartilage destruction, boneerosion, and bone formation. Each parameter was graded using thefollowing disease scale: 0=no disease; 1=minimal, 2=mild, 3=moderate,4=severe. In addition paws were assessed using visual disease severityscore (DSS), which measures swelling and redness on a scale of 0 to 3,with 0 being a normal paw, 1 being inflammation of one finger on thepaw, 2 being inflammation of two fingers or the palm of that paw, and 3being inflammation of the palm and finger(s) of the paw. Scores of 2 and3 are referred to herein as severely or highly inflamed paws.

Results are presented at FIGS. 3A-3C. Each data point represents onepaw, rather than an average for all four paws for an animal or anaverage over all animals. Reduction in the number of paws showing highpathology scores was statistically significant by three measures ofpathology (visual DSS—paw swelling and redness, cartilage damage andbone erosion) with higher anti-IL-17A 1D10 concentrations tested (28 and7 mg/kg). Results with the lowest concentration (2 mg/kg) werestatistically significant for bone erosion and reduced for visual DSSand cartilage damage. Similar benefits were observed in reduction ofproduction of cartilage degradative enzymes within inflamed paws (matrixmetalloproteases MMP-2, MMP-3, MMP-13).

Visual evaluation of paw inflammation, however, may underestimate thetherapeutic benefit of anti-IL-17A treatment of CIA mice, e.g. decreasedbone erosion. In other experiments, highly inflamed paws (DSS scores of2 or 3) from CIA mice were analyzed for bone erosion usinghistopathology or micro-computed tomography (micro-CT). This study waspossible because even though anti-IL-17A-treated animals had drasticallyreduced percentages of highly inflamed paws (see, e.g., FIG. 3A), thereremained a number of highly inflamed paws, and it was possible tocompare highly inflamed paws (DSS=2 or 3) from all treatment groups,including the no-antibody controls. FIG. 3D shows a plot of bone erosionfor highly inflamed paws from diluent treated, isotype control (rIgG1)treated, and anti-IL-17A antibody treated animals. Bone erosion, asmeasured by histopathology, was significantly reduced in paws fromanimals treated with anti-IL-17A when compared with no-antibodycontrols, despite their similar DSS scores. The results suggest thatsparing of bone erosion may be achieved with anti-IL-17A treatment evenin paws where there is no apparent improvement in inflammation asmeasured by DSS score.

Similar results were obtained when micro-CT was used to measure bonemineral density (BMD) for joints in highly inflamed paws in CIA mice.Table 15 provides BMD for paws with disease severity scores of 0 or 3from CIA animals treated with either an anti-IL-17A antibody of thepresent invention (1D10) or an isotype control (25D2). Even for jointswith the same visual disease severity, 1D10 antibody treated had onlyapproximately half the decrease in bone mineral density observed withisotype control treated animals.

TABLE 15 Bone Density for Joints in CIA Mice Treatment DSS BMD (mg/cc)25D2 3 95 25D2 3 108 1D10 3 288 1D10 3 299 1D10 0 502 1D10 0 480

As with bone erosion, cartilage destruction and pannus formation(proliferation of the synovial lining forming excessive folds ofinflamed tissue) were also reduced in anti-hIL-17A (1D10)-treated CIAmice. Histopathology showed that anti-IL-17A antibody treatment not onlyreduced the number of paws showing severe pathology, but also reducedpathology in paws that appeared equally inflamed based on visualinspection (DSS scores of 2 and 3) when compared with diluent or isotypetreated controls.

The observation that treatment with anti-IL-17A antibodies significantlyreduced bone erosion in the CIA model of joint inflammation suggeststhat such therapy may be useful in preventing one of the mostdebilitating and irreversible effects of RA in humans. In addition, theobservation that bone erosion is reduced even in highly inflamed pawssuggests that simple visual assessment of joint inflammation may notaccurately measure therapeutic efficacy in the lab, or ultimately in theclinic. Measurement of bone erosion may be necessary to track theeffects of therapeutic treatments. Such methods include, but are notlimited to, standard 2-D X-ray detection, computed tomography (CT),magnetic resonance imaging (MRI), ultrasound (US), and scintigraphy.See, e.g., Guermazi et al. (2004) Semin. Musculoskelet. Radiol.8(4):269-285.

Example 17 BAL Neutrophil Recruitment Assay of Anti-IL-17A Antibodies

The ability of anti-IL-17A antibodies of the present invention to blockthe activity of IL-17A in vivo was assessed in a bronchoalveolar lavage(BAL) neutrophil recruitment assay. Briefly, at day −4, five week oldfemale BALB/cAnN mice (Taconic Farms, Germantown, N.Y., USA) weretreated with anti-IL-17A antibodies of the present invention, or anisotype control, by subcutaneous injection of 0, 10, 30, 40, 60, or 100μg of antibody per mouse. At day −1 the mice were stimulated by nasaladministration of 1 μg of rhIL-17A in 50 μl of PBS (or a PBS onlycontrol) under light isoflurane anesthesia.

At day 0 the level of neutrophils present in BAL fluid was determined asfollows. Mice were euthanized with CO₂ and blood samples were collected,from which the concentration of anti-IL-17A antibody was determined. Aneedle was inserted into the upper cervical trachea through atracheotomy and BAL fluid was collected by introduction and removal of0.3 ml of PBS three times. The BAL fluid was centrifuged (400×g for 10minutes at 4° C.) and the cell pellet was resuspended in PBS. Total cellcounts were determined in a hemocytometer using trypan blue solution.Differential cell counts were performed on cytospin preparations byWright-Giemsa staining (Sigma-Aldrich, St. Louis, Mo., USA), accordingto standard morphologic criteria with use of oil immersion microscopy(original magnification ×1000). Cell counts were carried out on 200 or300 cells (lymphocytes, monocytes, neutrophils, eosinophils) todetermine the percent BAL neutrophil.

Results are provided at FIG. 4. Data are provided for three anti-IL-17Aantibodies of the present invention (1D10, 16C10 and 4C3), as well ascontrols. The percentage of neutrophils in BAL fluid as a percentage ofall leukocytes for individual experimental animals is plotted as afunction of serum antibody concentration, with the left segment of theabscissa (“0” to “1”) representing controls, as indicated in the legend.The controls show that rhIL-17A stimulus induces significant neutrophilrecruitment that is not reduced by administration of an isotype controlantibody (dosed at the same levels as the anti-IL-17A antibodies). Incontrast, the anti-IL-17A data show a dose-dependent reduction inrhIL-17A-induced neutrophil recruitment, with neutrophil recruitmentessentially blocked at serum antibody concentrations above 40-60 μg/ml.

Example 18 Treatment of Rheumatoid Arthritis (RA) Using Anti-IL-17AAntibodies

Human subjects diagnosed with RA who have had an inadequate response toone or more DMARDs are selected for treatment with a humanizedanti-IL-17A antibody of the present invention. Subjects are maintainedon methotrexate (10 mg/week), and are optionally treated with prednisonefor two weeks. Subjects are also dosed monthly with 50 or 100 mg ofanti-IL-17A antibody, administered subcutaneously. Doses are adjustedfor specific subjects according to standard clinical criteria and on thebasis of clinical response.

Response to treatment is assessed by determining the ACR score, which isbased on criteria developed by the American College of Rheumatology. TheACR score is a composite score that integrates multiple clinicalparameters and radiographic scores, such as reduction in the number ofswollen and tender joints, patient global assessment, physician globalassessment, pain scale, self-assessed disability, and acute phasereactants (erythrocyte sedimentation rate or C-reactive protein). SeeFelson et al. (1995) Arthritis & Rheumatology 38; 727-735. A subject isconsidered to have improved if he or she exhibits a score of ACR20 orhigher at week 24 or treatment. In addition, the proportion of subjectsachieving various ACR scores (e.g. ACR20, ACR50 and ACR70) can be usedto compare treatment and placebo groups in clinical trials to assess theclinical efficacy of the humanized anti-IL-17A antibody of the presentinvention

Example 19 Epitope Determination

Amino acid residues critical to binding of the antibodies of the presentinvention, e.g. rat 16C10, were determined a follows.

A first set of experiments was based on the observation that ratantibody 16C10 was able to bind human IL-17A (hIL-17A) but was unable tobind mouse IL-17A (mIL-17A) or related cytokine human IL-17F. Each ofthese three proteins was linked to an N-terminal FLAG® peptide tag (seeresidues 1 to 9 of SEQ ID NO.: 42). In order to identify amino acidresidues critical for 16C10 binding, various peptide subsequences ofFLAG-tagged hIL-17A, mIL-17A and IL-17F were combined by mixingrestriction fragments of the relevant genes to form hybrid polypeptides.Binding of these hybrid polypeptides to antibody 16C10 was determined inan Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen,Packard BioScience, Wellesley, Mass., USA) to determine which segmentsof hIL-17A were critical to binding. Biotinylated antibody 16C10 wasbound to streptavidin donor beads, and hybrid polypeptides were bound toacceptor beads having anti-FLAG® antibodies (Packard BioScience). Donorand acceptor beads were mixed, illuminated at 680 nm, and emission wasmeasured at 520-620 nm. Binding was measured as enhanced fluorescence insamples containing hybrid polypeptides that bound to antibody 16C10because acceptor beads were held in close proximity to donor beads andemitted light when singlet oxygen from the exited donor bead diffused tothe acceptor bead.

The results showed that antibody 16C10 binds to hybrid polypeptidescomprising amino acid residues 50-132, 63-132, 1-87, 1-112 and 63-87from hIL-17A (SEQ ID NO:40), demonstrating that residues critical to16C10 binding are present in residues 63-87 of hIL-17A (PSVIWEAKCRHLGCINADG NVDYHM) (amino acid residues 63-87 of SEQ ID NO. 40). Allresidue numbering in this example is with reference to the sequence ofhIL-17A (SEQ ID NO.: 40). For example, mIL-17A and IL-17F polypeptidessubstituted with residues 63-87 of hIL-17A (SEQ ID NO.: 40) were able tobind antibody 16C10, whereas the intact mIL-17A and IL-17F were not.

Point mutations were also introduced into hIL-17A to determine whichamino acid residues were critical to antibody 16C10 binding. In oneexperiment, alanine-scanning mutagenesis was performed in which analanine codon was introduced in place of the native amino acid atseveral residues (45, 46, 51, 52, 54, 55, 56, 57, 58, 60, 61, 62, 67,68, 70, 72, 73, 78, 80, 82, 84, 85, 86, 88, 93, 94, 95, 100, 101, 102,105, 108, 110, 111, 113, 114). Mammalian expression plasmids with genesencoding the mutant forms of hIL-17A were transiently transfected intohuman embryonic kidney (HEK) 293T cells. The supernatants were analyzedfor FLAG® peptide tag quantification and for antibody 16C10 binding byAlphaScreen, as described supra. None of the single amino acid alaninesubstitutions significantly reduced binding of antibody 16C10. Otherpoint mutations were made in which human IL-17F or mouse IL-17A residueswere substituted at various positions within the 63-87 fragment, i.e.L74Q, G75R, V83E, Y85H. Although none of these individual pointmutations inhibited antibody 16C10 binding, an hIL-17A. having all fourchanges exhibited substantially decreased binding, confirming thatresidues in the 63-87 fragment of hIL-17A, and more specificallyresidues in the 74-85 fragment (LGCINADGNVDY) (amino acid residues 74-85of SEQ ID NO:40), are important for 16C10 binding.

TABLE 16 Sequence Identifiers SEQ ID NO: Description 1 hu 16C10 lightchain DNA with signal sequence 2 hu 16C10 light chain amino acid withsignal sequence 3 hu 16C10 heavy chain DNA with signal sequence 4 hu16C10 heavy chain amino acid with signal sequence 5 hu 16C10/4C3 lightchain variable domain 6 hu 16C10/4C3 heavy chain variable domain 7 rat16C10 light chain variable domain 8 rat 16C10/4C3 heavy chain variabledomain 9 chimeric 16C10 light chain 10 chimeric 16C10 heavy chain 11rat/hu 16C10/4C3 CDRL1 12 rat/hu 16C10/4C3 CDRL2 13 rat/hu 16C10/4C3CDRL3 14 rat/hu 16C10/4C3 CDRH1 15 rat 16C10/4C3 CDRH2 16 hu 16C10/4C3CDRH2 (N54Q) 17 rat 16C10 CDRH2 (N54N/Q/A) 18 rat 16C10/4C3 CDRH3 19 hu16C10/4C3 CDRH3 (M96A) 20 rat 16C10 CDRH3 (M96M/A/K, M100hM/F) 21 rat4C3 light chain variable domain 22 hu 30C10 light chain variable domain23 hu 30C10 heavy chain variable domain 24 rat 30C10 light chainvariable domain 25 rat 30C10 heavy chain variable domain 26 rat/hu 30C10CDRL1 27 rat/hu 30C10 CDRL2 28 rat/hu 30C10 CDRL3 29 rat/hu 30C10 CDRH130 rat/hu 30C10 CDRH2 31 rat/hu 30C10 CDRH3 32 rat 12E6 light chainvariable domain 33 rat 12E6 heavy chain variable domain 34 rat/hu 12E6CDRL1 35 rat/hu 12E6 CDRL2 36 rat/hu 12E6 CDRL3 37 rat/hu 12E6 CDRH1 38rat/hu 12E6 CDRH2 39 rat/hu 12E6 CDRH3 40 huIL-17A (native) 41 rhIL-17A42 FLAG-IL-17A 43 R&D IL-17A 44 rat 23E12 light chain variable domainDNA with signal sequence 45 rat 23E12 light chain variable domain aminoacid with signal sequence 46 rat 23E12 heavy chain variable domain DNAwith signal sequence 47 rat 23E12 heavy chain variable domain amino acidwith signal sequence 48 rat/hu 23E12 CDRL1 49 rat/hu 23E12 CDRL2 50rat/hu 23E12 CDRL3 51 rat/hu 23E12 CDRH1 52 rat/hu 23E12 CDRH2 53 rat/hu23E12 CDRH3 54 rat 1D10 light chain variable domain 55 rat 1D10 heavychain variable domain 56 rat 1D10 CDRL1 57 rat 1D10 CDRL2 58 rat 1D10CDRL3 59 rat 1D10 CDRH1 60 rat 1D10 CDRH2 61 rat 1D10 CDRH3 62 hu 16C10light chain DNA with signal sequence 63 hu 16C10 heavy chain DNA withsignal sequence

Citation of the above publications or documents is not intended as anadmission that any of the foregoing is pertinent prior art, nor does itconstitute any admission as to the contents or date of thesepublications or documents. All references cited herein are incorporatedby reference to the same extent as if each individual publication,patent application, or patent, was specifically and individuallyindicated to be incorporated by reference.

What is claimed is:
 1. An isolated nucleic acid encoding a bindingcompound that binds to human IL-17A, wherein the binding compoundcomprises (a) a light chain antibody variable region comprising the CDRsequences of SEQ ID NOs: 11, 12 and 13 and (b) a heavy chain antibodyvariable region comprising the CDR sequences of SEQ ID NOs: 14, 17 and20.
 2. The isolated nucleic acid of claim 1, wherein the heavy chainvariable region comprises the CDR sequences of SEQ ID NOs: 14, 16 and19.
 3. The nucleic acid of claim 2, wherein the amino acid sequence ofthe light chain variable region is SEQ ID NO: 5 and the amino acidsequence of the heavy chain variable region is SEQ ID NO:
 6. 4. Thenucleic acid of claim 1, comprising the sequences of SEQ ID NOs: 1 and3, or the sequences of SEQ ID NOs: 62 and
 63. 5. An expression vectorcomprising the nucleic acid of claim 3 operably linked to controlsequences that are recognized by a host cell when the host cell istransfected with the vector.
 6. The expression vector of claim 5,wherein the binding compound comprises residues 1-220 of SEQ ID NO:2 andresidues 1-454 of SEQ ID NO:4 expression vector has ATCC Accession No.PTA
 7675. 7. An isolated A host cell comprising the vector of claim 6.8. A method of producing a polypeptide comprising: culturing the hostcell of claim 7 in culture medium under conditions wherein the nucleicacid sequence is expressed, thereby producing polypeptides comprisingthe light and heavy chain variable regions; and recovering thepolypeptides from the host cell or culture medium.